Long peptides of 22-45 amino acid residues that induce and/or enhance antigen specific immune responses

ABSTRACT

The invention is concerned with epitopes derived from human papilloma virus, and peptides having a size of about 22–45 amino acid residues comprising minimal T cell epitopes. The invention further provides clinically relevant approaches for immunizing subjects against (Myco)bacterially and/or virally infected cells or tumor cells, and in particular against HPV. The invention demonstrates that peptide sequences of 22–35 amino acid residues in length can induce both peptide-specific CD8+ cytolytic cells and CD4+ T-helper cells. Moreover, the invention demonstrates that vaccination with 22–35 residue long peptides results in a more vigorous CD8+ cytolytic T-cell response than vaccination with peptides of the exact minimal CTL epitope length. The invention further demonstrates that the intrinsic capacity of certain minimal CTL epitopes which instead of activating cytolytic effector cells tolerize these cytolytic cells, can be overcome by use of these 22–35 amino acid long peptides. The invention further provides clinically relevant approaches for vaccination and/or treatment of subjects against HPV. The invention also provides methods and uses suited to treat subjects suffering from progressive lesions and/or cervical cancer.

FIELD OF THE INVENTION

The present invention relates generally to the field of medicine, andmore specifically to induction and/or enhancement of a T cell responsedirected towards an antigen, using peptides comprising specific epitopesfor said antigen.

BACKGROUND OF THE INVENTION

The invention is exemplified mainly by using HPV directed immunity as amodel. However, the invention should not be read as being limited to HPVbut rather as being relevant for a wide variety of immune related orrelatable diseases.

HPV infection is highly prevalent among young, sexually active male andfemale individuals. Large prospective studies showed that acquisition ofHPV from male partners is common, occurring in 40–60% of subjects duringa 3 year follow-up period (Koutsky et al., 1997, Ho et al., 1998,Marrazzo et al., 2000). Therefore, HPV is probably the most commonsexually transmitted disease.

Papillomaviruses of the high-risk types (e.g. HPV16, 18, 31, 33, and 45)are responsible for cervical cancer (Bosch et al., 1995, Zur Hausen,1996). Following infection of the basal epithelial cells, the immediateHPV early genes E1, E2, E5, E6 and E7 are expressed. The E1 and E2 genesregulate viral replication. Furthermore, the E2 protein controls theexpression of the E6 and E7 oncoproteins. The E6 protein of thehigh-risk HPV types specifically binds to p53 and targets its rapiddegradation through the ubiquitin pathway. P53 is involved in initiationof apoptosis and loss of this protein result in the prevention ofapoptosis (Scheffner et al., 1990). The E7 protein of high-risk typesbinds to pRB, which normally prevents cells from entering the cell cycleby inactivating E2F, a protein needed for cell cycle entry (Dyson et al.1989). E7 expression results in the failure of infected cells towithdraw from the cell cycle and differentiate. Prolonged and elevatedexpression of the E6 and E7 oncoproteins is tightly associated withHPV-induced dysplasia and transformation into cervical carcinoma. Theprotective role of the immune system in the defense against HPV-relateddiseases and HPV-induced cancer in humans is suggested by the fact thatcompared to normal controls, immunosuppressed renal transplant patientsand patients infected with HIV display a 17-fold greater incidence ofgenital HPV infection (Ho et al., 1994, Matorras et al. 1991, Halpert etal. 1986). The diminished capacity of immunosuppressed individuals toresolve HPV infection indirectly points towards a protective role of theimmune system early in infection. Evidence of protection against HPV viaimmunity against the early antigens E2, E6 and E7 comes from thecottontail rabbit papillomavirus model which is the major animal modelfor cancer-associated papillomaviruses. Vaccination with thenonstructural proteins E1 and E2 induces the regression of virus-inducedpapillomas whereas viral tumor growth is suppressed. Furthermore,rabbits vaccinated with the combination of the E1, E2, E6, and E7 geneswere completely protected against viral challenge (Han et al. 1999,Selvakumar et al. 1995). These data indicate that immunity against E2,E6, and E7 can be effective as immunoprophylaxis of papillomavirusinfection as well as therapeutically for HPV-induced lesions and cancer.

Considerable interest exists in the identification of epitopes involvedin the immune response to HPV16, given the possibility to incorporatethese as subunits into a vaccine or to use these epitopes to monitorvaccine induced immunity in vivo. Since most epithelial cells expressMHC class I but not class II, the attention has so far been focused onthe induction of tumoricidal HPV-specific CD8+ cytotoxic T lymphocytes(Melief et al., 2000; Ressing et al., 1995; Ressing et al., 2000;Ressing et al., 1996). HPV-specific CD8+ T-cell reactivity has beenfound in the peripheral blood of patients diagnosed with cervicalintraepithelial neoplasia grade III (CIN III) lesions or cervicalcarcinoma (Nimako et al., 1997; Ressing et al., 1996) and intumor-infiltrating T-cell populations isolated from patients withcervical cancer (Evans et al., 1997). Tumor-specific CD4+ T helper(“Th”) immunity is now also considered pivotal for the efficienteradication of solid tumors, despite the fact that most of these tumorsdo not express MHC class II (reviewed in Melief et al., 2000; Pardolland Topalian, 1998; Toes et al., 1999). Recent evidence indicates thatCD4+ tumor specific T-cells are required not only for optimal inductionof CD8+ tumor specific CTL but also for optimal exertion of localeffector cell function by these CTL (Ossendorp et al., 1998, Toes etal., 1999). For induction of MHC class I restricted tumor-specificimmunity, cross-presentation of antigens that have been captured byprofessional antigen presenting cells appears to play a dominant role.For proper induction of tumor-specific CTL by cross-primingtumor-specific CD4+ T cell help is required (Toes et al., 1999,Schoenberger et al., 1998). A positive role for HPV-specific Th-immunitywas suggested by the predomination of CD4+ T-cells in regressing genitalwarts (Coleman et al., 1994) as well as by the detection of delayed-typehypersensitivity responses to HPV16 E7 in the majority of subjects withspontaneous regressing CIN lesions (Hopfl et al., 2000). Furthermore,HPV16-specific CD4+ T-cells have been detected in the blood of patientswith persistent HPV infections, high-grade CIN lesions or cervicalcancer (de Gruijl et al., 1998). For the less prevalent oncogenic typesHPV59 and HPV68, HLA-DR4 restricted Th-cells were isolated from theT-cells that infiltrated a cervical cancer lesion (Hohn et al., 1999).In contrast, despite the presence of HPV16 in the majority ofprogressive CIN lesions and cervical cancers, no in depth information isavailable concerning HLA-restriction and epitope specificity ofHPV16-specific T-helper responses.

For a clinically relevant approach of immunizing subjects, for instanceagainst (Myco)bacterially and/or virally infected cells or tumor cells,and HPV in particular, it is preferred that both specific T-helper cellsand CTL are induced. We have already shown that immunization withminimal CTL epitopes results in protection against tumors in some models(Feltkamp et al. 1993, Kast et al. 1991) whereas, in other models, itcan lead to tolerance or functional deletion of virus- andtumor-specific CTL that when otherwise induced are protective (Toes etal. 1996ab). The occurrence of tolerance or functional deletiondecreases the effects of vaccination significantly. However, until thepresent invention there was no solution for this phenomenon. Epitopesinvolved with this effect were therefore not suitable for immunizationpurposes. Processing of exogenous antigens for presentation by MHC classI molecules by cross-priming as well as by other mechanisms is nowwidely recognized second pathway of processing for presentation by MHCclass I, next to the well known endogenous route (Jondal et al. 1996,Reimann et al. 1997). The normal outcome of antigen processing via thispathway is CTL tolerance, unless APC activation by CD4+ T-cells takesplace (Kurts et al., 1997). Furthermore, in several studies with murinevirus infections, a positive correlation was detected between thefrequency of CTL precursors and protective immunity (Sedlik et al. 2000,Fu et al., 1999). For an optimal induction of CTL, presentation of CTLepitopes preferably takes place at the surface of professional antigenpresenting cells (APC's) such as dendritic cells (Mellman e al. 1998,Rodriguez et al. 1999). Whereas minimal CTL- and Th-epitopes can,without the need of processing by professional antigen presenting cells,be presented to T-cells, proteins need to be taken up and processed foran optimal presentation of CTL and Th-epitopes in MHC class I and MHCclass II, respectively, can occur (Manca et al. 1994).

BRIEF DESCRIPTION OF THE INVENTION

The present invention is concerned with means and methods for improvingantigen specific immune responses. To this end the invention provides amethod for inducing and/or enhancing an antigen specific T cell responsecomprising providing a system capable of exhibiting said response with apeptide comprising a T cell epitope specific for said antigen, saidpeptide comprising 22–45 amino-acid residues. These peptides can besynthesized efficiently whereas they also allow efficient uptake bycells capable of processing said peptide and present processed epitopesin the context of MHC-I or MHC-II. Preferably, said peptide is processedby a professional antigen presenting cell. Antigen presenting cells andparticularly professional antigen presenting cells such as dendriticcells are very efficient in processing and presenting epitopes, whereasthey further comprise additional functionalities allowing efficientcommunication with T-cells which ultimately leads to improved inductionand/or enhancement of said antigen specific T cell response. Thus in apreferred embodiment a method of the invention is provided wherein saidpeptide comprises a sequence capable of activating an antigen presentingcell. By a sequence capable of activating an antigen presenting cell ismeant a sequence which is capable of at least partly activating anantigen presenting cell preferably a professional antigen presentingcell. Said activation preferably leads to presentation of at least oneepitope of said peptide at the surface of said APC. In a particularlypreferred embodiment said peptide comprises at least two T cell epitopesfor said antigen. The presence of two T cell epitopes for said antigenallows an even more efficient induction and/or enhancement of saidantigen specific T cell response. Preferably, at least one of saidepitopes comprises a T-helper cell epitope for said antigen or acytotoxic T lymphocyte (CTL) epitope for said antigen. Having at leastone or the other epitope present on the peptide is favorable. Efficientinduction and/or enhancement is achieved when said peptide comprises aT-helper activating sequence. By a T-helper activating sequence is meantherein a sequence capable of at least partly activating a T-helper cell.Said activation preferably leads to improved induction and/orenhancement of said antigen specific T cell response. In one embodimentsaid peptide comprises at least one T-helper cell epitope for saidantigen and at least one cytotoxic T lymphocyte (CTL) epitope for saidantigen. Surprisingly, it was found that with such peptides the problemof induction of partial) tolerance against epitopes on said peptide doesnot, or to a lesser extent, occur whereas induction and/or enhancementof a specific T cell response for said antigen is very efficient. Thusin a particularly preferred embodiment of the invention said peptidecomprises at least one T-helper cell epitope for said antigen and atleast one cytotoxic T lymphocyte (CTL) epitope for said antigen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An epitope for an antigen is capable of interacting with a T-cellreceptor specific for said antigen, said T-cell receptor being specificfor an MHC-I or MHC-II molecule presenting an antigen derived orderivable peptide comprising said epitope. Though said T-cell receptormust be capable of interacting with a peptide derived or derivable fromsaid antigen, said epitope can be present on a wide variety of differentcompounds. Generally an epitope for antigen is present on an immunogenicpart of said antigen, said part being at least 9 amino-acids long andcapable of being presented by an MHC-I or MHC-II molecule. With thecurrent technology, however, it is possible to provide epitopes that aregenerated in an entirely different way or comprising different matter.For instance, once a fragment of said antigen is known to comprise anepitope, peptides can be generated that are different in one or moreamino-acid positions. Subsequently the presence or absence of saidepitope can easily be verified by, for instance, an ELISPOT assay usinga T cell specific for said epitope. Epitopes for an antigen can bepresent on a variety of different molecules such as but not limited topeptides, modified peptides, peptidomimetics.

A peptide of the invention may comprise an epitope for any particularantigen. However, preferably said antigen comprises a (Myco)bacterialand/or viral protein or an immunogenic part, derivative and/or analoguethereof. In one aspect of the invention, said antigen comprises aMycobacterium protein or an immunogenic part, derivative and/or analoguethereof. In a preferred embodiment of the invention said antigencomprises hsp65 369–412 (Ottenhof et al., 1991; Charo et al., 2001).Hsp65 369–412 comprises an HLA-A*0201 epitope at position 369–377,recognized by cytotoxic T-cells, and a DR5 epitope at position 390–412,recognized by T-helper cells. In another preferred embodiment, saidantigen comprises a human papillomavirus (HPV) protein or an immunogenicpart, derivative and/or analogue thereof. An immunogenic part,derivative and/or analogue of a protein comprises the same immunogeniccapacity in kind not necessarily in amount as said protein itself. Aderivative of such a protein can be obtained by preferably conservativeamino acid substitution.

More preferably, said protein comprises E2, E6 and/or E7. Regions wereidentified in the E2, E6 and E7 sequence that were most immunogenic.Furthermore, a number of naturally processed Th-epitopes mapping in thisregion were identified. Methods included respectively short andlong-term PBMC cultures derived from healthy blood donors. The PBMCcultures were stimulated with peptides of 22–35 amino acid residues inlength. In parallel, the in vivo induced E2, E6 and E7-specificimmunity, as detected by IFNγ ELISPOT assays, was analyzed in healthysubjects as well as subjects diagnosed with HPV16+ lesions.

Pathogen and tumor-specific T helper immunity was found to play apivotal role in the natural and vaccine-induced immune defense againstviral infections and tumors. The Th response against the human papillomavirus type 16 (HPV16) E2, E6 and E7 proteins was investigated in detail.By means of short and long term PBMC cultures from HLA-typed healthydonors, we identified three separate regions in the E2 protein (E231–120; E2 151–195; E2 271–365), the C-terminal part of HPV16 E6 (E681–158) and the central part of HPV16 E7 (E7 31–77) as the majorimmunogenic region within these antigens. Furthermore, we mapped tendistinct Th-epitopes within these proteins (DR1/E2 351–365, DR2/E2316–330, DR2/E2 346–355, DR4/E2 51–70, E2 61–76, DQ6/E2 311–325, DR15/E750–62, DR3/E7 43–77, DQ2/E7 35–50, DR1/E6 127–142).

By employing the IFNγ ELISPOT analysis, we detected in the CD45RO+T-cell memory subset Th-immunity against HPV16 E2 and HPV16 E6 inhealthy individuals, suggesting E2 and E6 directed protective immunityagainst previous HPV infection. Furthermore, Th-immunity was detectedtowards EPV16 E7 in subjects with HPV16+ lesions. Several of theseresponses matched with the three E7-derived Th-epitopes defined in thisstudy. A number of other HPV16+ subjects did not display any E7-specifictype 1-cytokine producing T cell immunity, indicating failure of theimmune response.

We have demonstrated that long peptides containing both CTL andTh-epitopes do not only induce antigen specific TH-cells but can induceantigen-specific CTL as well. Such peptides can also enhance antigenspecific CTL responses. In contrast with immunization of C57/B16 micewith the minimal E1A-derived CTL epitope (SGPSNTPPEI) (SEQ. ID. NO. 1)which results in enhanced tumor outgrowth, immunization with the HPLCpurified 32 amino acid long peptides containing this minimal CTL epitopeof adenovirus E1A protein (RECNSSTDSCDSGPSNTPPEIHPVVRLCPIIKP) (SEQ. ID.NO. 2) resulted in protection against a challenge with adenovirus E1A+RAS transformed tumor cells. Furthermore, HPV16 specificH-2Db-restricted CTL were induced upon immunization with 35 amino acidresidue long peptides, containing the minimal 9 amino acid long CTLepitope RAHYNIVTF (SEQ. ID. NO. 3), as shown by the presence ofH-2Db-RAHYNIVTF (SEQ. ID. NO. 3) tetramer positive CD8+T-cells in a FACSanalysis of spleen cells. Notably, in a direct comparison the CTLresponses induced by the 35-mer peptide were by far stronger than CTLresponses induced by the minimal CTL epitope and this difference waseven more pronounced after specific activation of professional antigenpresenting cells that can take up, process and present epitopes in thelong peptide optimally. Furthermore, the present invention shows thatstimulation of CTL by said long peptides, in the presence of antigenpresenting cell activating agents, results in the complete eradicationof small tumor nodules. In contrast, said minimal CTL epitope is notcapable of inducing complete eradication of tumors in all mice.

Thus, vaccination with a long peptide of the invention circumvents theinduction of tolerance which can occur when minimal CTL epitope peptidesare used. A long peptide of the invention leads to a higher amount ofCTL, generally associated with better protection. Without being bound bytheory, at least one of two independent mechanisms account for this. Onemechanism is the physical linkage of T-helper epitopes and CTL epitopesin one peptide. Another mechanism is the preferential presentation of atleast one CTL- and/or T-helper-epitope present in a long peptide of theinvention by professional antigen presenting cells. Because of its size,which excludes direct binding of a peptide of the invention to MHC classI, said peptide needs to be taken up by professional APC (eg. DendriticCells) that are able to process exogenously derived antigens and presentpeptides in MHC class I. Other cells are far less capable of processingand subsequent presenting epitopes. Thus background presentation bynon-professional APC's is at least in part prevented. The effect ofpreferential targeting of a peptide of the invention to APC is enhancedwhen a long peptide of the invention is used together with a dendriticcell-activating agent, which is a preferred embodiment. In anotherpreferred embodiment said antigen comprises an auto-antigen, preferablya tumor cell specific auto-antigen or a functional part, derivativeand/or analogue thereof. The term “auto-antigen” refers to an antigenthat is encoded by the animal species the T-cell belongs to. It alsorefers to an antigen that arises or becomes expressed as a result ofaberrant expression, modification, folding of proteins of said animaland/or mutation of sequences in the DNA or RNA of a cell encoding aprotein of said animal. In a preferred embodiment said auto-antigencomprises a MAGE, CTA or PSA antigen. In a particularly preferredembodiment said peptide comprises an antigen specific T-cell epitopethat induces tolerance and/or functional deletion of antigen specificCTL and/or T-helper cells in said system.

It is well known in the art that antigen specific immune responses maybe strengthened by providing systems capable of exhibiting saidresponses with an adjuvant. Adjuvants are also useful in the presentinvention. In a preferred embodiment said adjuvant comprises an exosome,a dendritic cell, MPL (monophosphoryl lipid A), poly I:C, Ampligen (polyI: poly C₁₂ U) and/or CpG nucleic acid.

According to the present invention, the advantage of epitope-linkagelies in the increased chance for simultaneous presentation of both theMHC class I and class II restricted epitopes on the surface of a singleAPC, thereby facilitating the delivery of cognate T cell help to CTLpriming. Presentation by professional antigen presenting cells ispreferred, because said cells can take up antigen more efficiently ascompared to non-professional antigen presenting cells. Moreover, onlyprofessional antigen presenting cells are capable, in response toantigen uptake, of activating T helper cells. Said activation isimportant for an optimal immune response.

The level of the CTL response is dependent on activation of dendriticcells as shown in this invention. One of the mechanisms that result inthe high efficacy of our long peptide HPV16 E7-vaccine, is the fact thatthe natural HPV16 E7 sequence contains a T helper- and CTL epitope thatare physically linked to each other. To obtain long peptides containingTh-epitopes and CTL epitopes that are preferably presented byprofessional antigen presenting cells, one can link two or more T-helperand CTL epitopes that are not naturally occurring sequences but thatresult in a considerable sized peptide. These T-helper and CTL epitopescan be derived from two different antigens, are preferably from the sametumor cell or pathogen but may be unrelated. According to the invention,a T cell response directed towards a specific antigen can as well beinduced or enhanced with a long peptide of the invention comprising aTh-epitope which is unrelated to said antigen. APC can generally beactivated by said unrelated Th-epitope. However, preferably saidTh-epitope is related to said antigen.

There are many systems available in which antigen specific immuneresponses can be induced and/or enhanced by providing said system with apeptide comprising an epitope specific for said antigen. For instance,in vitro cultures of peripheral blood mononuclear cells. Many othersystems are available. However, in preferred embodiment of the inventionsaid system comprises an animal. More preferably, said animal comprisesa human.

A method of the invention is very suited for providing an animal withimmunity against said antigen and/or for enhancing said immunity.Methods of the invention are suitable for any purpose that otherimmunization strategies are used for. Of old immunizations are used forvaccination purposes, i.e. for the prevention of disease. However,methods of the invention are not only suitable for preventing disease.Methods can also be used to treat existing disease, of course with thelimitations that the disease is treatable by inducing and/or enhancingantigen specific T cell immunity. This feature can be used to treat, forinstance, diseases associated with viral infections, such as somecancers. In principle, any disease curable in an immune related fashioncan profit from methods of the invention In a preferred embodiment saidanimal is suffering from or at risk of suffering from a disease that isat least in part treatable or preventable by inducing and/or enhancingsaid immune response. Preferably said disease comprises a viral diseaseand/or cancer. In another preferred embodiment said disease comprises a(myco)bacterial infection.

In another aspect the invention provides a peptide comprising 22–45amino acid residues, comprising a T cell epitope specific for anantigen. Preferably, said peptide comprises at least two T cell epitopesspecific for said antigen. Preferably said peptide comprises a CTLepitope for said antigen or a T helper cell epitope for said antigen. Ina particularly preferred embodiment said peptide comprises a CTL epitopefor said antigen and a T helper cell epitope for said antigen.Preferably said CTL and/or T-helper epitope for said antigen leads totolerance or functional deletion of CTL and/or T-helper cells,respectively, specific for said epitope, when present on a peptidecomprising 21 amino acid residues or less. In a preferred embodimentsaid peptide comprises between 22–35 amino acid residues. Morepreferably, said peptide comprises between 32–35 amino acids.

The invention further provides a use of a peptide of the invention forinducing and/or enhancing an immune response specific for said antigen.Provided is also the use of a peptide for the preparation of a vaccine.Preferably, for the preparation of a vaccine for the treatment and/orthe prevention of an HPV-related disease and/or cancer. Further providedis a use for the preparation of a medicament. Preferably for thepreparation of a medicament for the treatment of an individual sufferingfrom or at risk of suffering from a viral disease and/or cancer. Inanother preferred embodiment said individual is suffering from or atrisk of suffering from a (myco)bacterial disease.

In yet another aspect the invention provides a method of the inventionfurther comprising obtaining an antigen specific T cell from saidsystem. Thus also provided is an isolated T cell obtainable by a methodof the invention. Said T-cell can of course also be used for preparing amedicament. Provided is also a vaccine comprising a peptide of theinvention. Provided is also a medicament comprising a peptide or aT-cell of the invention.

Peptides of the invention can also be used to determine whether acollection of cells comprises an antigen specific T cell, said peptidecomprising an epitope for said antigen. For instance for determiningwhether an individual comprises immunity for said antigen. The presenceof said T cell is, preferably determined by ELISA, ELISPOT, delayed typehypersensitivity response, intracellular cytokine staining, and/orextracellular cytokine staining.

In another aspect the invention provides a peptide comprising animmunogenic epitope selected from an immunogenic region of humanpapilloma virus type 16 E2, E6 and E7 proteins. Preferably, saidimmunogenic regions comprises three separate regions in the E2 protein(E2₃₁₋₁₂₀; E2₁₅₁₋₁₉₅; E2₂₇₁₋₃₆₅) spanning amino acid 31–120, 151–195 and271–365 of human papillomavirus 16 E2 protein, the C-terminal region ofHPV16 E6 (E6₈₁₋₁₅₈) spanning amino acid region 81–158 in the HPV 16 E6protein and the central part of HPV16 E7 (E7₃₁₋₇₇) spanning amino acid31–77 of a human papilloma virus 16 E7 protein.

More preferably said immunogenic regions comprises in said E2 protein aregion spanning amino acid 46–75, a region spanning amino acid 51–70, aregion spanning amino acid 61–76, a region spanning amino acid 316–330,a regions spanning amino acid 311–325, a region spanning amino acid346–355 and a region spanning amino acid 351–365. In addition saidimmunogenic regions comprises in said E6 protein a region spanning aminoacid 81–158. More preferably said immunogenic regions comprises in saidE6 protein a region spanning amino acid 121–142, and a region spanning127–140. In addition said immunogenic regions comprises in said E7protein a region spanning amino acid 35–50, a region spanning amino acid50–62 and/or a region spanning amino acid 43–77. A T-helper cellresponse can be very well elicited and/or enhanced, using anyone of theabove-mentioned immunogenic regions. Notably, these immunogenic regionsare able to elicit/enhance T-helper responses restricted by differentHLA class II molecules.

In another aspect the invention provides a method to induce and/orenhance a T cell response directed towards an antigen, comprisingcontacting a T cell with a peptide, having a size of about 22–45 aminoacid residues, which comprises a minimal T cell epitope of said antigen.Preferably, a peptide of the invention has a size of about 22–40 aminoacid residues. Said minimal T cell epitope is preferably presented inthe form of a peptide having a size of about 22–40 amino acid residues,because we found that the issue of tolerance or functional deletion ofCTL by immunization with minimal epitopes can be circumvented by the useof long peptides (22–45; preferably 22–40 amino acid residues) which areable to prime CTL and/or T-helper cells. Said long peptides, comprisingat least one minimal T cell epitope, are capable of inducing and/orenhancing a T cell response. Immunization with said long peptide,comprising a minimal T cell epitope, results in protection even whensaid minimal epitope leads to tolerance or functional deletion of virus-and tumor specific CTL when administered in a bare form to said CTL.Said minimal T cell epitope may be derived from an immunogenic region ofthe invention. Thus, in one aspect, the invention provides a methodaccording to the invention, wherein said minimal epitope leads totolerance or functional deletion of virus- and tumor-specific CTL whenadministered in a bare form to said CTL.

In another aspect the invention provides a method to induce and/orenhance a T cell response directed towards an antigen, comprisingcontacting said T cell with a peptide, having a size of about 22–45amino acid residues, which comprises a minimal T cell epitope of saidantigen. Said minimal T cell epitope is presented in the form of apeptide having a size of about 22–45, preferably of about 22–40, aminoacid residues because we found that the induction of (virus- andtumor-specific) CTL is more efficient with said long peptides andresults in higher numbers of antigen-specific CTL. The use of said longpeptides results in a more efficient presentation of the comprised CTLand/or Th-epitopes. Without being bound by theory it is thought thatlong peptides need to be processed and presented by professional antigenpresenting cells. Supporting evidence for this theory comes from a moreefficient induction of CTL immunity after vaccination with said longpeptides and professional antigen presenting cells-activating agents.

Contacting a T cell with a peptide, in terms of the invention, ispreferably performed by means of a MHC class I or MHC class II molecule.Said MHC molecule may present said peptide after processing of thepeptide to said T cell. An antigen of the invention can be an antigenderived from a virus, a bacterium or associated with tumor cells.Preferably an antigen comprises a protein of human papillomavirus (HPV).More preferably, said HPV comprises HPV 16. In a most preferred aspect,said protein comprises HPV 16 E2, E6 and/or E7. Said long peptides arecapable of priming CTL and/or T-helper cells. Therefore, in one aspectthe invention provides a method according to the invention, wherein saidT cell comprises a CD8+ CTL. Alternatively, said T cell comprises a CD4+T helper cell.

By inducing a T cell response is meant herein that a T cell responsedirected towards a certain antigen is elicited. Before said induction,said T cell response was not present, or below detection levels or notfunctional By enhancing a T cell response is meant herein that theoverall action of T cells directed towards a certain antigen is madehigher and/or more efficient compared to the overall action of said Tcells before said enhancement. For instance, after said enhancement moreT cells directed towards said antigen may be generated. As a result, theaction of the additionally generated T cells increases the overallaction against said antigen. Alternatively, said enhancement maycomprise the increment of the action of T cells directed towards saidantigen. Said T cells may for instance react stronger and/or quickerwith said antigen. Of course, the result of said enhancement may begeneration of additional T cells together with increment of the actionof said T cells. Alternatively, said enhancement may comprise generationof additional T cells, or increment of the action of T cells, only.

A minimal T cell epitope is defined herein as a peptide derived from anantigen, capable of inducing a T cell response against said antigen.Said minimal T cell epitope is suitable to be bound by a class I or IIMajor Histocompatability molecule. Typically, a minimal CTL epitopeconsists of 8–11 amino acids. A minimal T cell epitope which is to bebound by an MHC class II molecule preferably at least consists of 11amino acids. In terms of the invention, administering a minimal epitopein a bare form means that said epitope is administered as a peptidehaving the size of said epitope, or as a peptide comprising said epitopeflanked on one or both sides with additional sequences, the size of saidpeptide being less than 22 amino acid residues. For instance, saidepitope may be flanked by processing sites.

With the teachings of the present invention it is possible to induceand/or enhance a T cell response directed towards an antigen using apeptide of the invention. Of course, examples of said peptide are alsoherewith provided. Thus, one embodiment of the invention provides apeptide, having a size of about 22–45, preferably of about 22–40 aminoacid residues, comprising at least one minimal T cell epitope. Saidpeptide comprises a minimal CTL epitope, and/or a minimal T helper cellepitope. Preferably said peptide comprises an immunogenic epitope of theinvention.

A peptide of the invention is more suitable to be used for vaccinationpurposes than a minimal epitope in a bare form, if immunization withsaid minimal epitope leads to tolerance or functional deletion of CTL.Thus in one aspect the invention provides a peptide according to theinvention, wherein said minimal CTL epitope leads to tolerance orfunctional deletion of virus- and tumor-specific CTL when administeredin a bare form to said CTL. A peptide of the invention preferably has asize of about 22–45 amino acid residues. Preferably, a peptide of theinvention has a size of 22–35 amino acid residues. More preferably, apeptide of the invention comprises 32 amino acid residues. In anotheraspect a peptide of the invention comprises a size of 35 amino acidresidues.

A peptide of the invention is more suitable to be used for vaccinationpurposes than a minimal epitope in a bare form, if immunization with apeptide of the invention leads to higher numbers of CTL. This can beachieved by assuring that presentation predominantly takes place byprofessional antigen presenting cells. Thus in one aspect the inventionprovides a peptide according to the invention wherein said peptide leadsto higher numbers of virus- and/or tumor-specific CTL than when minimalCTL epitopes are administered. In another aspect a peptide of theinvention leads to a vigorous CTL response capable of eradicating(small) tumor nodules.

The art currently knows many ways of generating a peptide. The inventionis not limited to any form of generated peptide as long as the generatedpeptide comprises a minimal T cell epitope. By way of example, a peptideof the invention can be obtained from protein E2, E6 or E7, synthesizedin vitro or by a cell, for instance through an encoding nucleic acid. Apeptide of the invention can be present as a single peptide orincorporated into a fusion protein. In one embodiment said peptide isflanked by processing sites allowing processing of said peptide inside acell such as to allow transport and/or incorporation into an MHCmolecule on the surface of said cell. In a preferred embodiment apeptide of the invention is after processing capable of complexing withan MHC class II molecule. MHC class II restricted T-cell immunity iscurrently considered to be important in eradication of for instancetumor cells although said tumor cells often do not express MHC class IImolecules. Peptides of the invention are particularly well suited foreliciting, inducing and/or stimulating both MHC class I and MHC class IIdependent T cells. Thus, in one aspect the invention provides a use of apeptide of the invention to induce and/or enhance a T cell responsedirected towards an antigen. A method to elicit and/or enhance an immuneresponse in an individual, comprising administering to said individual apeptide of the invention is also herewith provided. In another aspect,the invention provides a use of a peptide of the invention for thepreparation of a vaccine. Preferably, said vaccine comprises a vaccinefor, at least in part, prophylaxis of an HPV-related disease.

For instance, reactive T cells to HPV E2 and E6 protein have been foundin healthy individuals indicating that the identified immunogenicregions in the HPV16 E2 and E6 protein comprises epitopes relevant forprophylactic anticancer treatments based on vaccination and/or adoptiveimmunotherapy approaches. Additionally, reactive T cells to HPV E7 andHPV E6 protein have been found in subjects comprising CIN III lesionsand in subjects having cervical cancer. This indicates that theidentified immunogenic regions of the HPV16 E7 and E6 protein compriseepitopes relevant for anticancer treatments based on vaccination and/oradoptive immunotherapy approaches.

Peptides of the invention are suited for the generation and/or inductionof HPV specific T-cells. In one aspect the invention therefore providesa method for generating a human papilloma virus 16 specific T-cellcomprising contacting a collection of naïve T-cells with a peptide ofthe invention and culturing at least part of said collection of T-cells.In another aspect the invention provides a method for inducingantigen-specific cytokine (like, for instance, IL2 or IFNγ) productionby a human papilloma virus 16 specific memory T-cell comprisingcontacting a collection of T-cells of a subject infected with humanpapilloma virus 16 with a peptide of the invention and culturing atleast part of said collection of T-cells. In a preferred embodiment ofthese aspects said human papilloma virus 16 specific T-cell is isolated.

In yet another aspect a method of the invention further comprisesobtaining a T cell directed towards an antigen, comprising inducingand/or enhancing a T cell with a method of the invention and collectingformed T cells directed towards said antigen. Of course, an isolated Tcell obtainable by a method of the invention is also provided herein.Said T cell can be used in many ways, for instance for the detection ofan epitope of the invention for instance in a diagnostic assay. However,preferably said T cell is used in immunotherapy approaches. Suchapproaches incorporate but are not limited to approaches wherein such Tcells are infused into a subject suffering from an HPV-16 inducedcervical cancer or lesion. Thus in one aspect the invention provides ause of a T cell of the invention for at least in part treatment of anHPV-related disease. In such approaches it is preferred that said T celland said subject are histocompatible. Although this may not always betrue. In some cases mismatches for histocompatible even enhance thefunctionality of said T cell in said subject. The risk of developinggraft versus host disease can often be limited by assuring that T cellsnot restricted to an epitope of the invention are not present in a graftgiven to said subject. Of course, host versus graft responses resultingin elimination of infused T cells of the invention can occur. To limitsuch responses histocompatibility is preferred. However, in case whereineradication of tumor cells is very rapid, host mediated removal ofgrafted cells can be an advantage as an additional safety feature ofimmunotherapy. In one embodiment the invention therefore provides amethod for eliciting and/or enhancing an HPV16 E2, E6 and/or E7 proteinspecific immune response in an individual comprising administering tosaid individual a peptide of the invention or a T-cell of the invention.A peptide of the invention is particularly suited to elicit HPV 16specific immune responses in an individual. These immune responses canbe sufficiently high to provide a subject with protection againstinfection by HPV-16. However, a peptide or a T cell of the invention canalso be used to aid in combatting an already present infection in asubject. Thus another embodiment of the invention provides a use of apeptide or a T cell of the invention for the preparation of amedicament. Preferably, said medicament comprises a medicament for anHPV-related disease. Of course, a vaccine and/or a medicament comprisinga peptide of the invention is also provided herewith. Said vaccine isparticularly suitable for prophylaxis of an HPV related disease.Therefore the invention also provides a method for, at least in part,prophylaxis of an HPV-related disease, comprising administering to anindividual a vaccine of the invention. A medicament of the invention isof course particularly suitable for treatment of an HPV related disease.Thus, a method for, at least in part, treatment of an HPV-relateddisease, comprising administering to an individual a medicament of theinvention is also provided herewith. In another aspect the inventionprovides a method for determining whether a collection of T cellscomprises a memory T cell specific for an epitope comprising providingsaid collection of T cells with a peptide of the invention comprisingsaid epitope, culturing said collection of T cells in the presence of anantibody specifically directed towards an antigen-specific cytoline(like, for instance, IL 2 or IFNγ) and detecting any boundantigen-specific cytoline. In one embodiment said antibody is bound to asolid phase, preferably a solid surface. Said method is very useful todetermine whether an individual has a T cell response directed towards acertain antigen, for instance towards an HPV protein. Said method can beused for diagnosis of an HPV infection. Alternatively, said method issuitable to determine whether a T cell response is elicited aftervaccination of an individual. Preferably, said individual is providedwith a vaccine of the invention. Thus one embodiment of the inventionprovides a use of a peptide of the invention to determine the presenceof a T cell directed towards an antigen. In one embodiment, saidpresence of said T cell is determined in a sample. For instance, a bloodsample from an individual can be obtained. After that, the presence of aT cell directed towards a specific antigen can be determined using apeptide of the invention, in an immunologic assay. For instance, theabove mentioned method can be used. In this method, the detection ofbound antigen-specific cytokine (like, for instance, IL-2 or IFNγ)indicates the presence of said T cell Of course, a person skilled in theart can think of many more techniques for detection of a T cell directedtowards a certain antigen, using a peptide of the invention. Forinstance ELISA, ELISPOT, delayed type hypersensitivity (DTH) response,intracellular cytokine staining and extracellular cytokine staining orthe use of tetrameric MHC class II molecules comprising said peptidesare important techniques, known by the artisan.

The invention is further explained by the use of the followingillustrative examples.

EXAMPLES Example 1

Material and Methods

Subjects and Controls.

Samples of umbilical cord blood mononuclear cells (CBC) were used asimmunologically naive controls for influenza matrix-specific responses.PBMC of HLA-typed, anonymous healthy blood bank donors (D) obtainedafter informed consent, served as control PBMC for HPV16 E7 andinfluenza matrix-specific responses. Since these donors are anonymous,no additional data is available. However, donors with a known recenthistory of infection, including abnormal pap-smear, were, as part ofnormal regulations, discouraged to donate blood. The study of subjects(S; Table I) with CIN or cervical carcinoma in this paper was nested inthe “CIRCLE study” that investigates cellular immunity against HPV16infected cervical lesions. Women presenting with histologically provenCIN III or cervical carcinoma at the department of gynecology of theLeiden University Medical Center (LUMC) were, after informed consent,enrolled in this study. The study design was approved by the ethicscommittee of the LUMC. Blood was drawn at day of treatment. Subjectswith CIN III were treated by LEEP or cold knife conization. In case ofstage IB-IIA a radical hysterectomy was performed. All individuals forwhom enough PBMC were available were typed for HLA MHC class II (Naipalet al., 1984). Subjects were typed for HPV16 using HPV16-specificprimers on DNA isolated from paraffin-embedded sections of biopsies orsurgical resection specimens (Claas et al., 1989). Since HPV-specificT-helper response were expected to be found in subjects with progressivedisease (de Gruijl et al., 1998), we chose to analyze three subjectspresenting with CIN III, 4 subjects with stage IB cervical cancer and 4subjects with recurrent cervical cancer.

Antigens.

The peptides used spanning the E7 protein consisted of nine overlapping22-mer peptides and are indicated by their first and last amino acid inthe protein (1–22, 11–32, 21–42, 31–52, 41–62, 51–72, 61–82, 71–92 and77–98) or 4 long peptides defined by amino acids 1–35, 22–56, 43–77 and64–98. The peptides spanning the influenza matrix 1 protein of A/PR/8/43that were used as control peptides in the ELISPOT assay consisted ofsixteen 30-mer peptides overlapping by 15 amino acids. Peptides weresynthesized and dissolved as described previously (van der Burg et al.,1999).

Recombinant HPV16-E7 protein, HPV16-E6 protein and HIV-1 RT protein (thelatter two proteins served as control proteins in proliferation assays)were produced in recombinant E. coli transformed with Pet-19b-HPV16-E7,Pet-19b-HPV16-E6 (De Bruijn et al., 1998) or Pet-19b-HIV-1 reversetranscriptase RT) and purified as described previously (van der Burg etal., 1999).

Memory Response Mix (MRM): A mixture of tetanus toxoid (1 LF/ml;National Institute of Public Health and the Environment, Bilthoven, TheNetherlands) and Mycobacterium tuberculosis sonicate (2.5 μg/ml;generously donated by Dr. P. Klatser, Royal Tropical Institute, TheNetherlands) was used to control the capacity of PBMC to proliferate inresponse to typical recall antigens.

HLA-DR-peptide Binding Assay.

Binding of peptides was measured as reported previously (van der Burg etal., 1999). Briefly, as a source of HLA-DR molecules B-lymphoblasticcell lines homozygous for HLA-DR were used: LG2.1 (DRB*0101, DR1), IWB(DRB1*0201, DR2), HAR (DRB*0301, DR3), BSM (DRB*0401, DR4) and Pitout(DRB1*0701, DR7). DR molecules were purified by affinity chromatographyand the purity confirmed by SDS-PAGE. The analysis of peptide binding topurified HLA-DR molecules was performed using N-terminallyfluorescence-labeled standard peptides. As standard fluorescent peptidesin the binding assays, either HA 308–319 (PKYVKQNTLKLAT, DR1 and DR2)(SEQ. ID. NO. 4), hsp65 3–13 (KTIAYDEEARR, DR3) (SEQ. ID. NO. 5),HA₃₀₈-319 Y→F (PKFVKQNTLKLAT, DR4) (SEQ. ID. NO. 6) or Ii 80–103(LPKPPKPVSKMRMATPLLMQA LPM, DR7) (SEQ. ID. NO. 7) were used.

Immunogenicity Assay.

HPV16-E7 derived peptide induced proliferation of PBMC isolated fromblood obtained from healthy donors was measured as described previously(van der Burg et al., 1999). Briefly, PBMC were seeded at a density of1.5×10⁵ cells/well of a 96 well U-bottom plate (Costar, Cambridge,Mass.) in 200 μl of ISCOVE's medium (Gibco) enriched with 10% autologousserum, in the presence or absence of 10 μg/ml of indicated 22-mer E7peptide. As positive control, PBMC were cultured in the presence of aMemory Response Mix. Peptide specific proliferation was measured at day6 by tritium-thymidine incorporation. Peptides were scored asimmunogenic, i.e., able to stimulate T-cells, when the proliferationof >25% of the 8 test wells exceeded the mean proliferation+2 times thestandard deviation of medium control wells.

Proliferation Assays.

Cultures were pulsed with 0.5 μCi [³H] thymidine (5 Ci/mM, Amersham, UK)per well for 18 hours. Plates were harvested with a Micro cell Harvester(Skatron, Norway). Filters were packed in plastic bags containing 10 mlof scintillation fluid and subsequently counted in a 1205 Betaplatecounter (Wallac, Turku, Finland). MHC class II blocking experiments werecarried out as described before using murine monoclonal antibodiesagainst HLA-DQ SPV.L3, against HLA-DR B8.11.2 and against HLA-DP B7/21(van der Burg et al., 1999). Antibodies were added to APC 1 h prior toprotein-APC incubation.

Isolation and Expansion of HPV16-E7-specific T-helper Cells.

Peptide-specific T-cell bulk cultures were generated as describedpreviously (van der Burg et al., 1999) using either the E7 22-mer or E735-mer peptides. Specific proliferation was measured by incubation of50,000 responders with an equal amount of irradiated (30Gy) APC(autologous PBMC unless indicated otherwise) and peptide or protein asindicated. E7-peptide and -protein-specific bulk T-cells were cloned bylimiting dilution as described previously (van der Burg et al;, 1999).

Cytokine Assays.

To determine specific excretion of cytokines, T-cell clones werestimulated by incubation of 50,000 T-cells with an equal amount of APC(30Gy) together with 10 μg/ml peptide, control peptide, E7 protein orcontrol protein as indicated. After 24 hours of incubation, supernatantwas harvested and replicate wells were pooled. Cytokine production wasmeasured by Enzyme-Linked Immunosorbent Assay (ELISA) as describedpreviously (van der Burg et al., 1999).

Analysis of Antigen-specific T-cells by ELISPOT.

PBMC or cord blood cells (CBC) were seeded at a density of 2×10⁶cells/well of a 24 well plate (Costar, Cambridge, Mass.) in 1 ml ofISCOVE's medium (Gibco) enriched with 10% FCS, in the presence orabsence of 5 μg/ml of indicated E7-derived 22-mer peptide. As positivecontrol PBMC were cultured in the presence of indicated pools ofinfluenza A/PR/8/34 M1 protein derived peptides consisting of 4overlapping 30 amino acid long peptides in each pool. Based on ourobservations, we used a 4-day stimulation before PBMC were transferredto the ELISPOT plates. This resulted in a pronounced IFNγ-productiontowards influenza M1-derived peptides in the CD45RO+ (memory) subset ofT-cells but not in naïve T-cells obtained from adult PBMC (unpublishedobservations). Following 4 days of incubation at 37° C., PBMC wereharvested, washed and seeded in six replicate wells at a density of 10⁵cells/well of a Multiscreen 96-well plate (Millipore, Etten-Leur, TheNetherlands) coated with a IFNγ catching antibody (Mabtech A B, Nacha,Sweden). The ELISPOT was further performed according to the instructionsof the manufacturer (Mabtech). The number of spots were analyzed with afully automated computer assisted video imaging analysis system (CarlZeiss Vision). Specific spots were calculated by subtracting the meannumber of spots+2×SD of the medium only control from the mean number ofspots of experimental wells. Antigen-specific T-cell frequencies wereconsidered to be increased compared to non-responders when T-cellfrequencies were> 1/10,000 PBMC.

Results

Identification of the Immunogenic Sequences Within HPV16 E7.

We set out to identify the sequences within the HPV16 E7 protein thatfunction as major immunogenic determinants in the context of MHC classII. A set of HPV16 E7 derived overlapping peptides was tested forbinding to HLA-DR 1, 2, 3, 4 and 7 in a quantitative peptide/MHC bindingassay (Geluk et al., 1995). Together, these HLA-DR molecules cover atleast 50–60% of the caucausian, oriental and negroid populations (Bauret al., 1984). Four peptides, E7₁₋₂₂, E7₄₁₋₆₂, E7₅₁₋₇₂ and E7₇₇₋₉₈,bound to three or more different HLA-DR molecules (Table II): peptideE7₁₋₂₂ bound to HLA DR2, 3, 4 and 7, peptide E7₄₁₋₆₂ bound to DR1, 2, 3and 4 whereas peptide E7₅₁₋₇₂ bound to DR1, 3 and 7 and peptide E7₇₇₋₉₈bound to DR1, 2 and DR7. This is in accordance with the fact that thepeptide binding restrictions for MHC class II molecules were found to beless strict than those for MHC class I (Rammensee et al., 1995). Ofnote, these peptides failed to bind to one or two other DR moleculestested indicating that their binding, although rather ubiquitous incharacter, was specific.

Our sequent experiments focused on the four peptides binding to multipleHLA-DR molecules, as these are the most likely to comprise naturallypresented T-helper (Th) epitopes (Geluk et al., 1998). We analyzedwhether the peptides could stimulate T-cells to proliferate by addingthese to PBMC from 13 HLA-typed healthy blood donors. This assay doesnot discriminate between the reactivity of memory T-cells and naïve, invitro, primed T-cells but can be readily employed for identification ofimmunogenic peptides (van der Burg et al., 1999). All four peptides wereable to stimulate proliferative responses in PBMC from multiple donors(Table II). Most PBMC cultures reacted to the peptides E7₄₁₋₆₂ andE7₅₁₋₇₂ indicating that the central region of the E7 protein harbors themost immunogenic sequences and is likely to be targeted by the immunesystem in HPV16 infected individuals.

TABLE I Subject characteristics Patient HPV number Age type MXC class IItype Stage of Disease 1 38 16 HLA DR4, 11(5);DQ3 CIN III 2 41 16 HLADR15(2), 3;DQ6(1), 2 CIN III 3 32 16 HLA DR15(2), 4;DQ3, 6 CIN II 4 6716 HLA DR15(2), 4; FIGO IB DQ6(1), 8(3) 5 59 16 HLA DR11(5);DQ3 FIGO IB6 36 16 Non-Available FIGO IB 7 28 16 HLA DR1,7;DQ5(1), 2 FIGO IB 8 4816 HLA DR15(2), 7; DQ6(1) Recurrent cervical carcinoma 9 46 16 HLADR15(2), 7; DQ6(1), 2 Recurrent cervical carcinoma 10 47 16 HLADR6,7;DQ1, 2 Recurrent cervical carcinoma 11 46 16 HLA DR3, DQ2Recurrent cervical carcinoma

TABLE II Binding capacity and immunogenicity of HPV16-E7 derivedpeptides Immunogenicity D #1 D #2 D #3 D #4 D #5 E7 Binding DR1,3 DR1,4DR1,4 DR3,6 DR2,3 aa DR1 DR2 DR3 DR4 DR7 DQ1,2 DQ5,8 DQ6,8 DQ6,2 DQ1,2 1–22^(a)  >70^(b) 10 0.8 30 7 −^(c) − − − + 11–32 >70 >70 50 >70 >7021–42 >70 12 >70 >70 >70 31–52 >70 >70 >70 >70 NT 41–62     7.4 50 5538 >70 − − + − + 51–72   70 >70 50 >70 11 − − − − + 61–82 >70 1565 >70 >70 71–92   12 2 >70 >70 >70 77–98   46 22 >70 >70 0.9 + − − − −Memory Response Mix + + + + + Immunogenicity D #6 D #7 D #8 D #9 D #10 D#11 D #12 D #13 E7 DR2,3 DR1,3 DR2,13 DR4,7 DR1,3 DR7 DR3,13 DR7,13 aaDQ6,2 DQ5,2 DQ6 DQ3 DQ5,2 DQ2 DQ6,2 DQ6,3  1–22^(a) + − − − − − − +11–32 21–42 31–52 41–62 + + + + + − − + 51–72 + + + + + − − + 61–8271–92 77–98 − − + + + − − + Memory + + + + + + + + Response Mix^(a)First and last amino acid number of 22-mer overlapping peptidesderived from the HPV16-E7 protein. ^(b)Binding capacity of each peptideis expressed as the IC50 value: this is the test peptide concentration(μM) at which 50% of the maximal binding of the standard fluorescencelabeled peptide is inhibited. >70, indicates undetectable binding.^(c)Fresh PBMC derived from HLA-DR and -DQ typed healthy blood donors(D) were stimulated with peptides that bound to ≧3 differentHLA-purified DR molecules. Donors were considered to react specificallyto a peptide when >25% of all wells tested displayed a proliferationthat exceeded the mean +2x standard deviation of the medium onlycontrol. More then 85% of all wells stimulated with the Memory ResponseMix were found positive.

TABLE III Evaluation of HPV16 E7-specific T cell responses by IFNγELISPOT CBC #1 CBC #2 CBC #3 D #20 D #21 D #22 D #23 D #24 D #25 D #26 S#1 HLA- NA NA NA 3^(b) 15(2) 1 2 4 1 11(5) 4 DR 13(6) 13(6) 4 8 7 11(5)HLA- NA NA NA 1, 2 6(1) 3, 5 1 4, 8 5 NA 3 DQ E7  1–22^(a) <1 1 <1 <1 <1<1 <1 <1 11–32 <1 1 <1 15 <1 <1 <1 <1 21–42 <1 <1 2 <1 <1 <1 <1 <1 31–52<1 <1 <1 <1 <1 <1 1 <1 41–62 <1 2 2 <1 <1 <1 1 <1 51–72 <1 <1 4 <1 <1 <11 32 61–82 <1 <1 <1 <1 <1 <1 2 <1 71–92 <1 <1 <1 <1 <1 <1 2 <1 77–98 <1<1 <1 <1 <1 <1 1 <1 M1 pool 1  <1^(c) <1 <1 8 9 46 <1 7 5 13 <1 pool 2<1 <1 <1 12 36 41 <1 28 <1 65 8 pool 3 <1 <1 <1 <1 47 42 <1 <1 9 17 92pool 4 <1 <1 <1 <1 47 38 27 <1 15 2 <1 S #2 S #3 S #4 S #5 S #6 S #7 S#8 S #9 S #10 S #11 HLA- 15(2) 15(2) 15(2) 11(5) NA 1 15(2) 15(2) 6 3 DR3 4 4 7 13(6) 7 7 HLA- 6(1), 2 6(1), 3 6(1), 8(3) 3 NA 5(1), 2 6(1)6(1), 2 1, 2 2 DQ E7  1–22^(a) <1 <1 <1 16 <1 <1 <1 <1 <1 <1 11–32 <1 <1<1 <1 <1 <1 <1 <1 8 21–42 <1 <1 <1 4 <1 <1 1 <1 <1 2 31–52 8 <1 <1 <1 <1<1 23 <1 <1 14 41–62 14 <1 <1 <1 <1 <1 19 <1 <1 5 51–72 76 <1 <1 98 <1<1 <1 <1 <1 <1 61–82 <1 <1 <1 <1 <1 <1 <1 <1 <1 13 71–92 <1 <1 <1 <1 <1<1 <1 <1 <1 <1 77–98 <1 <1 <1 12 <1 <1 6 <1 <1 6 M1 pool 1 <1 27 <1 <1 1113 <1 <1 <1 15 pool 2 <1 29 47 2 8 66 11 <1 <1 19 pool 3 6 <1 59 100 <141 39 <1 <1 20 pool 4 22 15 <1 10 <1 34 17 <1 21 28 ^(a)First and lastamino acid number of 22-amino acid long peptides derived from theHPV16-E7 protein that were used to stimulate T-cells of umbilical cordblood cells(CBC), PBMC from donors(D) or PBMC from HPV16+ subjects(S).For the influenza M1 protein only the number of the pool is indicated.Each pool consists of four 30 amino acid long peptides that overlap by15. ^(b)HLA class II typing of each individual. NA, non available^(c)The number of antigen-speciific cells per 100,000 cord blood cellsor PBMC. Frequencies ≧1/10,000 are depicted in bold. Underscored are theresponses that match in specificity to that of Th-clones described inFIGS. 1, 2, 3.Mapping of Naturally Processed T-helper Epitopes in HPV16 E7.

As a next step in the identification of Th-epitopes in HPV16 E7, peptidespecific bulk T-cell cultures were generated from PBMC of HLA-typedhealthy blood donors by repeated stimulation with several long peptidescovering the central region of E7. The responding T-cells weresubsequently tested for recognition of antigen presenting cells (APC)pulsed with either peptide or whole E7 protein. In the latter setting,presentation of the relevant peptide-epitopes requires antigen-uptakeand -processing. Approximately 30% of the Th-cell cultures generatedspecifically responded to the peptides against which they were raised.Of these, three cultures also showed modest but specific activityagainst E7 protein pulsed APC, indicating the presence of Th-cells withthe capacity to respond against naturally processed antigen (FIGS. 1 a,2 a and 3 a). FIG. 1 b shows the reactivity of a Th-clone that wasisolated from HLA-DR15, 4 and DQ6, 7 positive PBMC raised againstpeptide E7₄₁₋₆₂. These Th-cells specifically respond to APC pulsed withpeptide E7₄₁₋₆₂, peptide E7₄₃₋₇₇ or E7 protein. The specificity of theresponse by this Th-clone exceeded that of the polyclonal culture fromwhich this Th-clone was isolated (compare to FIG. 1 a), illustratingthat by limiting dilution we have succeeded in isolating the T-cells ofinterest. Further studies using MHC class II blocking antibodies againstHLA-DR, -DQ or -DP (FIG. 1 b) and partially MHC class II matched APCrevealed that the Th-clone was restricted by HLA-DR15 (FIG. 1 c). Finemapping of the epitope showed that the core sequence recognized wasE7₅₀₋₆₂. Furthermore, this Th-clone produced the Th type 1 cytokine IFNγ(FIG. 1 d). In a similar fashion a HPV16 E7-specific Th-clone wasobtained from HLA-DR3, DQ2 positive PBMC stimulated with peptide E7₂₂₋₅₆(FIG. 2 a). Also this Th-clone secreted IFNγ upon triggering by peptideE7₂₂₋₅₆ and E7 protein pulsed APC (FIG. 2 bc). Further analysis showedthat this Th-clone recognized the core sequence E7₃₅₋₅₀, as indicatedthe fact that both peptides E7₃₀₋₆₀ and E7₃₅₋₅₅ recognized, in anHLA-DQ2 restricted fashion (FIG. 2 c). A third type 1 cytokine producingTh-clone with distinct specificity was derived from a HLA-DR1, 3 and DQ2positive PBMC culture stimulated with peptide E7₄₃₋₇₇ (FIG. 3 ab). Thisclone was HLA-DR3 restricted (FIG. 3 cd) and produced IFNγ uponrecognition of both peptide and protein pulsed APC (FIG. 3 e). Whereaspeptide E7₄₃₋₇₇ was recognized, the smaller peptide E7₄₁₋₆₂ was not,indicating that the C-terminal part of the E7₄₃₋₇₇ peptide harbors thecore epitope. In conclusion, using established Th-clones we mapped threenaturally processed epitopes in HPV16 E7. It is conceivable that HPV16+individuals expressing the relevant MHC class II molecules display invivo induced Th-immunity against these peptides.

Memory T-helper Responses in Subjects with HPV16+ CIN III Lesions orCervical Carcinoma.

In an approach parallel to the mapping of Th-epitopes using PBMCcultures of healthy individuals, the response against HPV16 E7 insubjects with HPV16+ lesions was analyzed (see Table I for subjectcharacteristics). Primary, in vitro, stimulation of naive T-cells fromnewborns or adults can result in the production of IL-2 andproliferation of T-cells. However, at this stage such T-cells fail tosecrete IFNγ. The production of IFNγ follows when the antigen isencountered again (Early and Reen, 1999; Pittet et al., 1999; Sallustoet al., 1999). This feature allowed us to discriminate between in vitroprimed and memory T-cell responses by ELISPOT. Th-responses against theinfluenza A matrix (M1) protein, which are readily detectable in PBMC ofmost donors of diverse HLA-types (Table III and our unpublishedobservations), were measured in parallel as a positive control for thedetection of memory T-cell immunity in the assay. Stimulation withM1-peptides of MACS-separated CD45RA+ (naïve) T-cells and CD45RO+(memory) T-cells resulted in the production of IFNγ in the CD45RO+subset only, confirming that our ELISPOT setup specifically detectsmemory responses (FIG. 4). Furthermore, cord blood cells (CBC), inaccordance with their naive phenotype, did not react by secreting IFNγwhen stimulated in vitro with influenza M1 derived peptides. In additionto the PBMC from all healthy donors, those from 9/11 of the HPV16+subjects reacted to one or more pools of M1-peptides (Table III).

Analysis of HPV16 E7-immunity revealed responses against one or morepeptides in 2/3 subjects diagnosed with CIN III and in 3/8 subjects withcervical carcinoma. In addition, one of the seven donors testeddisplayed immunity against a peptide. The central region of HPV16 E7that is covered by the peptides E7₃₁₋₅₂, E7₄₁₋₆₂ and E7₆₁₋₇₂ wastargeted by the immune system of all five HPV16+ individuals (TableIII), not only confirming that this region is highly immunogenic (TableII) but also demonstrating that this region harbors epitopes that aretargeted by natural immune responses against HPV16. Interestingly, thespecificity of IFNγ responses detected in three HPV16+ subjects, incombination with their HLA-type, matched that of the establishedTh-clones for which we examined specificity and HLA-restriction indetail (FIGS. 1–3). In particular (Table III, underscored frequencies),subject 2 exhibited significantly increased immunity matching thespecificity of the two HLA-DR restricted clones as well as weak immunitymatching the HLA-DQ2/E7₃₅₋₅₀ clone. Subject 8 displayed a responsematching the DR15/E7₅₀₋₆₂ clone, whereas subject 11 showed reactivitymatching the HLA-DQ2/E7₃₅₋₅₀ clone. It should be noted that in othersubjects expressing the relevant MHC class II molecules such responsescould not be detected (subjects 3, 4, 7 and 10).

Example 2

Materials and Methods

Lymphocytes

Peripheral blood mononuclear cells (PBMC) and serum of HLA-typedanonymous healthy blood donors were obtained after informed consent.Because these donors are anonymous, no data on medical history areavailable. Importantly, donors with a known recent history of infection,including abnormal pap-smear were, as part of normal regulations,discouraged to donate blood.

Antigens

A set of peptides spanning the whole HPV16 E2 protein consisting of 23overlapping peptides, 22 of which have a length of 30 amino acids andone of which (E2₃₃₁₋₃₆₅) has a length of 35 amino acids, was used. Thesepeptides share an overlap of 15 amino acids. For epitope fine-mappingand the determination of cross-reactivity of HPV16-E2 specificTh-clones, peptides with a length of 15 and 20 amino acids were used.The peptides spanning the Influenza Matrix 1 protein of A/PR/8/34, whichwere used as control peptides in ELISPOT assays, consisted of 16 30-merpeptides overlapping by 15 amino acids. Peptides were synthesized bysolid phase technique on an automated multiple peptide synthesizer(Abimed AMS 422, Langenfeld, Germany), and analysed by reverse phaseHPLC. The lyophilized peptides were dissolved in 50 μl of DMSO, dilutedin PBS to a final concentration of 2.5 mg/ml. The HPV16 E2 C-terminal(E2₂₈₀₋₃₆₅) protein and HPV16 E7 protein were produced according topreviously described procedures (Franken et al., 2000)

Memory Response Mix (MRM), consisting of a mixture of tetanus toxoid(0.75 LF/ml final concentration; National Institute of Public Health andEnvironment, Bilthoven, The Netherlands), Mycobacterium tuberculosissonicate (2.5 μg/ml; generously donated by Dr. P. Klatser, RoyalTropical institute, Amsterdam, The Netherlands) and Candida albicans(0.005%, HAL Allergenen Lab, Haarlem, The Netherlands), was used toconfirm the capacity of PBMC to proliferate and produce cytokine inresponse to common recall antigens.

HLA-DR—Peptide Binding Assay

Binding of peptides to HLA-DR was measured as reported previously (vander Burg et al., 1999). Briefly, as a source of DR molecules B-LCLhomozygous for DR were used: LG2.1. (DRB*0101, DR1), IWB (DRB1*0201,DR2), HAR (DRB*0301, DR3), and BSM (DRB*0401, DR4). DR molecules werepurified by affinity chromatography and the purity confirmed bySDS-PAGE. The analysis of peptide binding to purified DR molecules wasperformed using N-terminally fluorescence-labeled standard peptides. Asstandard peptide in the binding assays HA₃₀₈₋₃₁₉ (PKYVKQNTLKLAT, DR1 andDR2), (SEQ. ID. NO. 4), hsp65 3–13 (KTIAYDEEARR, DR3) (SEQ. ID. NO. 5)or HA₃₀₉₋₃₁₉ Y→F (PKFVKQNTLKLAT) (SEQ. ID. NO. 6) was used.

Short-Term T Cell Proliferation Assay

Immunogenicity of individual HPV16 E2 peptides was determined by shortterm proliferation assays of healthy donor PBMC with HPV16-E2 peptides,according to previously described procedures (van der Burg et al. 1999).Briefly, freshly isolated PBMC were seeded at a density of 1.5×10⁵cells/well in a 96-well U-bottom plate (Costar, Cambridge, Mass.) in 200μl of IMDM (Iscove's Modified Dulbecco's Medium, Bio Whittaker,Verviers, Belgium) supplemented with 10% autologous serum (10% FCS wasused for CBC cultures). HPV16-E2 peptides were added at a concentrationof 10 μg/ml. Medium alone was taken along as negative control;phytohemagglutinine PHA, 0.5 μg/ml) served as a positive control. Foreach peptide 8 parallel micro-cultures were initiated; each donor wastested twice. Peptide-specific proliferation was measured at day 6 by³H-thymidine incorporation. Peptides were scored positive, when—in bothassays—the proliferation of >50% of the test wells exceeded the meanproliferation+3×SD of the control wells, and the stimulation index (SI)of all test wells over medium control wells was higher than 3.

Generation and Analysis of Long-term HPV16-E2 Specific Th-cultures

Long-term HPV16 E2-specific T-cell cultures and Th-clones wereestablished according to previously described procedures (van der Burget al. 1999). Briefly, PBMC from healthy HLA-typed donors werestimulated in vitro with the following HPV16 E2 peptides(E2₂₇₁₋₃₀₀+E2₂₈₆₋₃₁₅; E2₃₀₁₋₃₃₀; E2₃₁₆₋₃₄₅; E2₃₃₁₋₃₆₅). 15×10⁶ PBMC wereseeded in 25 cm² culture flasks (Nalge Nunc, USA) in 6 ml IMDMsupplemented with 10% autologous serum. Peptides were added at aconcentration of 5 μg/mL At day 7, 15×10⁶ PBMC were added, together withfresh medium and peptides. At day 14 and 21 viable T cells wereharvested from the cultures, counted and restimulated with an equalamount of autologous irradiated PBMC and peptide (5 μg/ml). T-cellgrowth factor (Biotest, Dreieich, Germany) was added 2 days afterrestimulation at a final concentration of 10%. The T-cell cultures weretested for peptide recognition by proliferation assay at day 28.Peptide-specific T-cell cultures were cloned by limiting dilution andT-cell clones were subsequently tested for the recognition of E2-peptideand -protein-pulsed APC. Specificity of the Th clones was analysed asdescribed previously (Van der Burg et al. 1999). Notably, inproliferation assays in which Th-clones were tested for proteinrecognition, autologous monocytes were used as APC. For measurement ofproliferation, cultures were pulsed with 0.5 Ci [³H] thymidine (5μCi/mM, Amersham, UK) per well for 18 hours. Plates were harvested witha Micro cell Harvester (Skatron, Norway). Filters were packed in plasticcovers containing 10 ml of scintillation fluid and subsequently countedon a 1205 Betaplate counter (Wallac, Turku, Finland). HLA-class IIblocking experiments were performed using murine monoclonal antibodies:anti-DQ SPV. L3, anti-DR B8.11.2, and anti-DP B7/21. Supernatants of theproliferation assays were harvested 24 hrs after incubation and analysedfor the presence of IFN-γ by ELISA (van der Burg et al., 1999).

Detection of Memory Th-cells by ELISPOT

Memory cells (CD45RO⁺) were isolated freshly from buffycoats by MACSafter incubation with CD45RO microbeads (cat. no. 460-01, MiltenyiBiotec, Germany). The purity of the obtained CD45RO+ fraction was >95%as determined by flowcytometry after surface staining for CD45RO andCD45RA (CD45RA-FITC, cat. no. 347723, CD45RO-PE, cat. no. 347967, BectonDickinson Biosciences, USA). CD45RO+ cells were seeded at a density of10⁶ cells/well in a 24-wells plate (Costar) in 1 ml of IMDM supplementedwith 10% FCS. 10⁶ irradiated autologous cells were added to each well asAPC. The responder cells were incubated with either medium alone, poolsof HPV16-E2 peptides at 5 μg/ml/peptide, MRM 1:50 dilution or pools ofInfluenza Matrix peptides (positive controls) and cultured for 11 daysin order to improve the detection of antigen-specific cells (Mc Cutcheonet al. 1997). The cells were then harvested, washed and seeded in 4replicate wells at a density of 5×10⁴ cells/well of a Multiscreen96-well plate (Millipore, Etten-Leur, The Netherlands) coated with aIFN-γ catching antibody. Per well, 10⁵ irradiated autologous PBMC wereadded as APC together with 5 μg/ml peptide. ELISPOT analysis was furtherperformed according to the instructions of the manufacturer (Mabtech AB, Natcha, Sweden). Analysis of the number of spots was done with afully automated computer-assisted-video-imaging analysis system (CarlZeiss Vision).

Intracellular Cytokine Staining (ICS) of Memory T-cells

Autologous monocytes were isolated from PBMC by adherence to aflat-bottom 48-wells plate during 2 hours in X-vivo 15 medium (BioWhittaker, Verviers, Belgium) at 37° C., and then used as APC. CD45RO+cells were stimulated for 11 days with peptide, then harvested, washedand suspended in IMDM+0.1% BSA at a concentration of 1.5×10⁶ cells/ml.200 μl of cell suspension was added to the monocytes +200 μl of 10 μg/mlHPV16-E2 peptide (stimulated) or 200 μl of medium (non-stimulatedcontrol). After 1 hour of incubation at 37° C., 800 μl of IMDM+10%FCS+12.5 μg/ml Brefeldin A (Sigma) was added and cells were incubatedfor another 5 hours. The cells were then harvested, transferred into aV-bottom 96-wells plate, washed twice with ice-cold PBS and fixed with50 μl paraformaldehyde 4% for 4 minutes on ice. Following fixation, thecells were washed once with cold PBS and once with PBS/NaAz 0.2%/BSA0.5%/Saponin 0.1%. This was followed by an incubation in 50 μl PBS/NaAz0.2%/BSA 0.5%/Saponin 0.1%/FCS 10% for 10 minutes on ice. Cells werewashed twice. with PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1% and supernatantwas removed before 25 μl of PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1%containing 1 μl FITC-labelled mouse-anti-human IFN-γ (0.5 g/ml, BDPharmingen, cat. no. 554551) 2 μl PE-labelled anti-CD4 (BD Bioscience,cat. no. 345769), and 2 μl PerCP-labelled anti-CD8(BD Bioscience cat.no. 347314) was added. Following 30 minutes of incubation at 4° C., thecells were washed, suspended in 100 μl paraformaldehyde and analysed byflowcytometry.

Homology Search in Protein Database

The search for sequence homology of the overlapping HPV16 E2 peptides ina protein database (SwissProt) was performed using standard Basic LocalAlignment Tool (BLAST: www.ncbi.nlm.gov/blast/blastcgi). Statisticalsignificance threshold (EXPECT) was 10 (Altschul et al., 1997). Reportedmatches with >60% aminoacid homology with HPV16 E2 peptides wereincluded.

Results

High Reactivity of Healthy Donor PBMC Against HPV16 E2-derived Peptides.

We examined the proliferative responses of healthy donor PBMC againstHPV16 E2 protein by using an array of overlapping 30-mer peptidescovering the entire E2 sequence. Incubation of freshly isolated PBMC of8 HLA-typed donors with each of the 23 E2-derived 30-mer peptides showedthat 4 out of 8 donors reacted to 2 or more of the peptides. Theobserved E2 peptide-specific proliferative responses were remarkablystrong (Table IV). In all cases more than 75% of the parallelmicrocultures reacted against the stimulating peptide. For instance, intwo independent experiments we found the peptide-specific proliferationof donor #8 against peptides E2₃₁₋₆₀, E2_(46-75,) E2₉₁₋₁₂₀, E2₁₅₁₋₁₈₀,E2₂₇₁₋₃₀₀ and E2₂₈₆₋₃₁₅ to exceeded background proliferation in 75–94%of all eight parallel microcultures tested. This points at the presenceof a very high frequency of E2-specific T-cells in the PBMC isolates. Inparticular, PBMC of donors #3, 5 and 8 displayed strong responses with abroad specificity. Please note that the 30-mer peptides have a 15 aminoacid overlap with their neighboring peptides. Consequently, responsesagainst adjacent peptides (e.g. donor #3, E2₃₁₋₆₀ and E2₄₆₋₇₅) mostlikely involve the same epitope, whereas responses against non-adjacentpeptides are directed against distinct epitopes.

Not only the frequency of responding cultures but also the magnitude ofthe proliferative responses were remarkably high. The peptide-specificproliferation of several cultures from donors #3, 5 and 8 exceededbackground with mean stimulation indices ranging from 9.2–16.5 and, assuch, are comparable to responses found against the tetanus toxoidantigen in several of the donors (#3, 4, 5 and 7; SI ranging from13.3–25; see Table IV). These stimulation indices clearly exceed thethreshold (SI≧3) that is commonly used for the detection of memory Tcell responses (Bermas et al., 1997). Please note that responses againsttetanus toxoid are considerably higher in some of the other donors(SI≧100 in donors #3 and 6), but that these strong values most likelyrepresent very broad responses against multiple epitopes comprised by anentire antigen, rather than against a single 30-mer E2-peptide. Takentogether, our data indicate that the T cell repertoire of healthy donorscan contain particularly high frequencies of T-cells specific for theHPV16 E2 antigen resulting in vigorous proliferative responses, andsuggest that these responses may reflect T-cell memory.

TABLE IV Immunogenicity of HPV16 E2 derived peptides. D1¹ D2 D3 D4 D5 D6D7 D8 E2_(1–30) ² E2_(16–45) E2_(31–60) 81% (8,8)³ 88% (8,6) E2_(46–75)88% (5,5) 81% (6,8) 94% (6,5) E2_(61–90) E2_(76–105) E2_(91–120) 94%(4,8) 75% (4,0) E2_(106–135) E2_(121–150) E2_(136–165) E2_(151–180) 81%(6,4) 81% (4,1) E2_(166–195) 94% (16,5) 81% (10,6) E2_(181–210)E2_(196–225) E2_(211–240) E2_(226–255) E2_(241–270) E2_(256–285)E2_(271–300) 88% (4,8) E2_(286–315) 88% (16,2) 94% (9,2) E2_(301–330)E2_(316–345) 94% (4,7) E2_(331–365) 100% (11,4) 75% (4,1) TT 100% (29,1)100% (56,5) 100% (25) 100% (16,4) 100% (23,5) 100% (139) 100% (13,3)100% (59) ¹D: Donor, Eight different healthy blood donors were tested.²HPV16 E2 peptides are indicated by the first and last amino acid. TT isthe common recall antigen tetanus toxoid. ³Peptides were scoredpositive, when - in both assays - the proliferation of >50% of the testwells exceeded the mean proliferation +3 * SD of the control wells, andthe stimulation index (SI) of the positive test wells over mediumcontrol wells was higher than 3. Only positive scores are depicted asthe mean percentage of wells exceeding medium control +3 * SD from thepercentages found in both assays. Between brackets the meanstimulationindex of all test wells of both assays.HPV16 E2-specific Th Cultures Recognize Naturally Processed Epitopes.

The proliferation data pointed at the existence of multiple immunogenicTh-epitopes within HPV16 E2. We performed a more detailed analysis ofthe nature and specificity of such responses, thereby focussing on theN-terminal and C-terminal region which our data (Table IV) revealed tocomprise several highly immunogenic peptides. The overlapping peptidescomprised in this C-terminal region (E2₂₇₁₋₈₀₀; E2₂₈₆₋₃₁₅; E2₃₀₁₋₃₃₀;E2₃₁₆₋₃₄₅; E2₃₃₁₋₃₆₅) were tested for their capacity to bind to HLA-DRmolecules. Each of the five peptides showed intermediate to strongbinding to two or more of the common HLA-DR molecules tested (Table V),which supports the notion that these peptides can indeed represent classII MHC-restricted Th-epitopes.

TABLE V Binding affinity of HPV16-E2 derived peptides to differentHLA-DR types Amino acid sequence HPV16-E2 DR1 DR2 DR3 DR4FNSSHKGRINCNSNTTPIVHLKGDANTLKC 271–300 22 65 37 >70TPIVHLKGDANTLKCLRYRFKKHCTLYTAV 286–315 8 68 20 >70LRYRFKKHCTLYTAVSSTWHWTGHNVKHKS 301–330 6 8 >70 10SSTWHWTGHNVKHKSAIVTLTYDSEWQRDQ 316–345 6 55 30 >70AIVTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSI 331–365 8 20 10 15 Binding affinityof each peptide is expressed as the IC₅₀ value: this is the peptideconcentration (μM) at which binding of the standard fluorescence labeledpeptide is reduced to 50% of its maximal value. >70 representsundetectable binding

Subsequently, long-term E2-specific Th cultures were generated throughstimulation of PBMC from HLA-typed healthy blood donors at weeklyintervals with either peptides E2₂₇₁₋₈₀₀ and E2₂₈₆₋₃₁₅, peptidesE2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅, or with peptide E2₃₃₁₋₃₆₅ or with peptideE2₄₆₋₇₅. PBMC from 2 donors showed strong peptide-specific proliferativeresponses against one or more of the stimulating peptides (data notshown). Through cloning via limiting dilution of these cultures, wesucceeded in the isolation of stable T cell clones, uniformly displayinga CD4⁺CD8⁻ phenotype, against six distinct peptide-epitopes. Two ofthese Th clones were established from HLA-DR15(2)-, -DQ6(1)- PBMCstimulated with E2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅. Although both Th-clonesrecognized E2₃₀₁₋₃₃₀, in depth analysis of the specificity of theseTh-clones revealed that they recognized distinct, yet overlapping,sequences, restricted by different class II HLA molecules. One of theclones recognized peptide E2₃₁₆₋₃₃₀ in the context of HLA-DR15(2),whereas the other clone was specific for peptide E2₃₁₁₋₃₂₅ in thecontext of HLA-DQ6(1) (FIGS. 5, 6). The two other Th-clones weresimilarly related, in that one recognized peptide E2₃₄₆₋₃₅₅ in thecontext of DR15(2), whereas the other reacted against E2₃₅₁₋₃₆₅ in aDR1-restricted manner (FIGS. 7, 8). Notably, these data are incorrespondence with the peptide binding data in Table V, in that longervariants of these DR15(2) and DR1-restricted epitopes were indeed foundto bind to the restricting HLA molecules (binding assay not availablefor HLA-DQ6). In addition, two clones were found to react to peptideE2₄₅₋₇₅, one reacted against peptide E2₅₁₋₇₀ in a HLA-DR4 restrictedmanner and the other clone reacted against peptide E2₆₁₋₇₅. (FIG. 15)

Further evidence that the four E2 peptides identified representphysiologically relevant Th epitopes is provided by the fact that theE2-specific Th clones did not only respond against peptide-loaded APC,but also specifically responded against APC that were pulsed with the E2protein (FIGS. 5–8). Because in the latter case presentation of thepeptide epitopes depends on uptake and processing of the E2 antigen, andnot merely on exogenous loading of class II molecules at the APC cellsurface, these data provide definite proof that the four E2 peptidesrecognized by our Th clones correspond to naturally processed epitopes.Finally, all four Th-clones produced IFN-γ upon antigenic stimulation,which is indicative of a Th-type 1 cytokine profile. Taken together ourdata show that the T cell repertoire of healthy individuals harborsIFN-γ-secreting E2-specific CD4⁺ Th cells (FIGS. 5–8).

Detection of HPV16 E2 Specific Memory Th-cells in Healthy Individuals

The strikingly frequent detection of HPV16 E2-specific Th immunity inhealthy individuals, as described in the first paragraph, prompted us toanalyse whether the underlying T cell repertoire would representimmunological memory as a the result of previous encounter with antigen,or whether it would primarily consist of particularly abundant naïve Tcell precursors specific for this antigen. In view of the high incidenceof, generally transient, genital HPV infections in young sexually activeindividuals (Karlsson et al., 1995; Koutsky et al., 1997; Ho et al.,1998), as well as the prominent expression of E2 during HPV infection,it seems conceivable that T cell memory against E2 is to be found inhealthy subjects. We examined the nature of the HPV16 E2-specificimmunity detected by us through analysis of the E2-specific reactivityof the CD45RO⁺ fraction of healthy donor PBMC, which containsantigen-experienced T-cells but is devoid of their naïve counterparts(Young et al., 1997). Because we found the T cell repertoire of healthydonors to contain IFNγ-producing Th cells (FIGS. 5–8), we measured theantigen-specific T-cell responses through IFNγ ELISPOT. We firstanalysed the reactivity of CD45RO⁺ T cells of two healthy donors whilefocusing our attention on a selection of E2 peptides which on basis ofour previous experiments (Table IV) appear to be localized in the mostimmunogenic regions of HPV16 E2. Interestingly, these CD45RO⁺ PBMC werefound to respond against multiple E2 peptides (FIGS. 9 a,b), supportingthe notion that healthy subjects can display HPV16E2-specific T cellmemory. We confirmed that the responding IFNγ-producing cells belongedto the CD4⁺ Th cell subset by employing IFNγ intracellular cytokinestaining instead of ELISPOT as a readout (not shown). A broader surveyof E2-specific reactivity against the full array of peptides, usingCD45RO⁺ PBMC from 8 additional healthy donors, revealed that 4 of thesePBMC isolates responded against one or more of the HPV16 E2 peptides(FIGS. 9 c–f and not shown). Taken together, our data reveal thepresence of CD45RO⁺ memory-type, IFNγ secreting Th cells reactiveagainst HPV16 E2 peptides in approximately half of the healthy donorstested. Notably, the incidence by which these responses are detected isvery similar to that of the strongest subset of proliferative responsesfound in total PBMC (Table IV), implicating that also these responsesare likely to represent reactivity by memory T cells rather than by invitro primed naïve T cells.

Cross-reactivity of HPV16 E2 Specific Th-clones with Peptide Sequencesof Other HPV Types

Due to the common nature of HPV infections, a majority of the humanpopulation is likely to encounter multiple HPV types (Thomas et al.2000; Koutsky et al. 1997). Furthermore, the protein sequences of theviral gene products are conserved to considerable extend between HPVtypes. It is therefore possible that at least a fraction of the T cellrepertoire induced by a previous encounter with a given type of HPVcould cross-react, and therefore cross-protect, during subsequentinfection with other HPV types. Alignment of the sequence of the HPV16E2 protein with that of various other HPV types revealed that it is mostprominently conserved with that of other high risk types. Although thisconservation is somewhat less conspicuous when the HPV16 E2 sequence iscompared to that of low risk or common types it is evident that in allcases maximal conservation is confined to certain regions within the E2sequence. In particular, three areas of HPV16E2 share homology with E2of other HPV types: the N-terminal portions E2₃₁₋₁₂₀ and E2₁₅₁₋₁₉₅ aswell as the C-terminal portion E2₂₇₁₋₃₆₅. These regions co-localise withthe major functional domains of E2, in that the N-terminal domainharbours the transcriptional activation functions of this proteinwhereas the C-terminal portion mediates its sequence-specificDNA-binding properties. The intervening sequences ranging from residues210 to 270 constitute the so-called hinge-region connecting the two keyfunctional domains, which is poorly conserved between HPV types.Interestingly, our analyses of E2-specfic responses in short-termproliferation assays have revealed that the most immunogenic peptidesare clustered in the two conserved domains of the HPV16 E2 sequence (seeTable IV). In view of these considerations, we tested whether ourestablished Th clones, raised against epitopes derived from theC-terminal part of the HPV16 E2 sequence (FIGS. 5–8), would be capableof cross-reacting with E2 peptides of a number of other HPV types thatshared maximal homology with HPV16 with respect to this particular E2sequence. Indeed, the DQ6-restricted E2₃₁₁₋₃₂₅-specific Th-clone showedstrong recognition of the corresponding peptides of HPV types 26, 31, 35and 45 (not shown). The amino acid homology within this epitope variesfrom 73 to 87% (identical or amino acids with similar physico-chemicalproperties). Our other Th clones did not reveal considerablecross-reactivity for highly homologous E2 peptides of other HPV types(not shown). These data indicate that part of the HPV16 E2-reactive Thmemory detected in our assays may relate to encounter of HPV types otherthan HPV16, but also suggest that the majority of this immune repertoirewas most likely established through encounter with HPV16 itself.

Example 3

Material and Methods

Subjects and Controls

PBMC of anonymous healthy blood bank donors (D) were obtained. Sincethese donors are anonymous, no additional data is available. However,donors with a known recent history of infection, including abnormalpap-smear, were, as part of normal regulations, discouraged to donateblood.

The study of subjects (S; Table II) with CIN or cervical carcinoma inthis paper was nested in the “CIRCLE study” that investigates cellularimmunity against HPV16 infected cervical lesions. Women presenting withhistologically proven CIN III or cervical carcinoma at the department ofgynecology of the Leiden University Medical Center (LUMC) were, afterinformed consent, enrolled in this study. The study design was approvedby the ethics committee of the LUMC. Blood was drawn at day oftreatment. Subjects with CIN III were treated by LEEP or cold knifeconization. In case of stage IB-IIA a radical hysterectomy wasperformed. Subjects were typed for HPV16 using HPV16-specific primers onDNA isolated from paraffin-embedded sections of biopsies or surgicalresection specimens (Claas et al., 1989). Since HPV-specific T-helperresponse were expected to be found in subjects with progressive disease(de Gruijl et al., 1998), we chose to analyze subjects with stage IBcervical cancer.

Antigens.

The peptides used spanning the E6 protein consisted of 15 overlapping22-mer peptides and are indicated by their first and last amino acid inthe protein (e.g. 1–22, 11–32, 21–42, 31–52, 41–62, 51–72, 61–82, 71–92and so forth, the last peptide consists of amino acid 137–158). Peptideswere synthesized and dissolved as described previously (van der Burg etal., 1999).

Memory Response Mix (MRM): A mixture of tetanus toxoid (1 LF/ml;National Institute of Public Health and the Environment, Bilthoven, TheNetherlands) and Mycobacterium tuberculosis sonicate (2.5 μg/mLgenerously donated by Dr. P. Klatser, Royal Tropical Institute, TheNetherlands) was used to control the capacity of PBMC to proliferate inresponse to typical recall antigens.

Analysis of Antigen-specific T-cells by ELISPOT.

PBMC were seeded at a density of 2×10⁶ cells/well of a 24 well plate(Costar, Cambridge, Mass.) in 1 ml of ISCOVE's medium (Gibco) enrichedwith 10% FCS, in the presence or absence of 5 μg/ml of indicatedE6-derived 22-mer peptide. As positive control PBMC were cultured in thepresence of indicated pools of influenza A/PR/8/34 M1 protein derivedpeptides consisting of 4 overlapping 30 amino acid long peptides in eachpool.

In case CD45RO+ cells were used the CD45RO+ cells were seeded at adensity of 10⁶ cells/well in a 24-wells plate (Costar) in 1 ml of IMDMsupplemented with 10% FCS together with indicated single or pools ofpeptides at a concentration of 10 μg/ml/peptide. Following 4 days ofincubation at 37° C., PBMC were harvested, washed and seeded in four tosix replicate wells at a density of 10⁵ cells/well of a Multiscreen96-well plate (Millipore, Etten-Leur, The Netherlands) coated with aIFNγ catching antibody (Mabtech A B, Nacha, Sweden). The ELISPOT wasfurther performed according to the instructions of the manufacturer(Mabtech). The number of spots were analyzed with a fully automatedcomputer assisted video imaging analysis system (Carl Zeiss Vision).Specific spots were calculated by subtracting the mean number ofspots+2×SD of the medium only control from the mean number of spots ofexperimental wells. Antigen-specific T-cell frequencies were consideredto be increased compared to non-responders when T-cell frequencies were=1/10,000 PBMC.

Isolation of the CD45RO+ Memory Fraction of PBMC

Memory cells (CD45RO⁺) were isolated freshly from buffycoats by MACSafter incubation with CD45RO microbeads (cat. no. 460-01, MiltenyiBiotec, Germany) according to the instructions of the manufacturer. Thepurity of the obtained CD45RO+ fraction was >95% as determined byflowcytometry after surface staining for CD45RO and CD45RA (CD45RA-FITC,cat. no. 347723, CD45RO-PE, cat. no. 347967, Becton DickinsonBiosciences, USA).

Results

In view of the high incidence of, generally transient, genital HPVinfections in young sexually active individuals (Karlsson et al., 1995;Koutsky et al., 1997; Ho et al., 1998), as well as the prominentexpression of HPV16 E2-specific immunity (see example 2) in about halfof the healthy subjects tested, we studied the natural in vivo induced,HPV16 E6-specific response in the human population by means of the 4-dayIFNγ-ELISPOT assay. This assay detects only memory T helper-cells, whichupon stimulation will secrete IFNγ whereas their naïve counterparts willnot (see example 1 and 2 and references therein). A survey ofE6-specific reactivity against the full array of peptides present inPBMC from 18 healthy blood donors, revealed that 11 of these 18 (>60%)PBMC isolates responded against one or more of the HPV16 E6 peptides(Table VIa). The majority of the responses were found at the C-terminalend of E6 (E6₈₁₋₁₅₈). Furthermore, all healthy donors reacted againstthe common recall antigen influenza matrix 1 protein. The frequency ofboth influenza M1 and HPV16 E6-specific T-helper cells were ofcomparable magnitude. Interestingly, a similar survey in HPV16+ patientsrevealed that only 3/12 (25%) reacted against HPV16 E6 peptidesindicating that the E6 response found in healthy subjects is protectiveagainst disease.

TABLE VI Detection of HPV16 E6-specific T-help r reactivity in healthydonor PBMC. Donor¹ 1–22² 11–32 21–42 31–52 41–62 51–72 61–82 71–9281–102 91–112  1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1  2 <1 <1 <1 <1 <1 <1 <1<1 <1 <1  3 <1 4 <1 3 <1 <1 <1 <1 <1 <1  4 <1 <1 <1 10 <1 <1 <1 <1 <1 <1 5 <1 5 1 1 2 9 5 7 6 10  6 3 34 <1 9 <1 <1 2 <1 11 14  7 <1 2 1 <1 <1<1 <1 <1 2 14  8 4 4 <1 <1 9 <1 <1 <1 12 14  9 2 <1 7 3 <1 <1 4 <1 <1 310 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 11 2 <1 <1 2 <1 <1 <1 7 3 4 12 <1 <1 <1<1 <1 <1 <1 <1 <1 <1 13 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 14 5 5 <1 10 5 <12 20 12 5 15 18 16 4 15 2 1 <1 3 12 21 16 <1 <1 <1 <1 <1 <1 <1 <1 <1 <117 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 18 <1 <1 <1 <1 4 <1 <1 <1 2 <1 101–111– 121– 131– 137– Donor¹ 122 132 142 152 158 M1P1 M1P2 M1P3 M1P4  1 <1<1 <1 <1 <1 <1 16 <1 <1  2 <1 <1 <1 <1 4 2 26 12 7  3 <1 3 8 18 4 30 719 <1  4 <1 <1 <1 <1 <1 22 15 14 10  5 2 8 5 8 1 7 10 12 37  6 6 12 10<1 6 <1 33 41 5  7 <1 <1 1 3 14 6 21 57 28  8 3 6 7 3 <1 27 38 34 16  9<1 <1 3 <1 <1 22 10 14 4 10 <1 <1 7 <1 <1 13 23 19 11 11 2 15 10 27 1733 28 24 52 12 <1 <1 14 15 <1 5 10 12 <1 13 <1 1 <1 <1 <1 <1 <1 1 21 1428 18 37 40 2 12 42 28 45 15 8 11 8 <1 <1 52 68 64 5 16 <1 <1 <1 <1 <139 66 49 48 17 <1 <1 <1 <1 <1 4 3 <1 66 18 <1 11 1 1 <1 <1 1 10 <1 ¹18different healthy blood donors were tested. ²First and last amino acidnumber of HPV16 E6-derived peptides are indicated. M1P1–M1P4 are thefour-different pools of 4, by 15 residues overlapping, 30-amino acidlong peptides derived from influenza A matrix 1 protein(inf A/PR/8/34).³Indicated are the number of specific spots per 100,000 PBMC. Specificspots are calculated by subtracting the mean number of spots + 2xSD ofthe medium control from the mean number of spots after stimulation withpeptide. Numbers in bold indicate T-cell frequencies ≧1/10,000

TABLE VIb Detection of HPV16 E6-specific T-helper reactivity in HPV16+patients. patient 1–22 11–32 21–42 31–52 41–62 51–72 61–82 71–92 81–10291–112 101–122 C40 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C41 <1 <1 <1 <1 <1<1 <1 <1 <1 <1 2 C43 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C47 <1 <1 <1 28 <1<1 <1 <1 <1 <1 <1 C57 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 2 C58 <1 <1 <1 <1 <1<1 <1 <1 <1 <1 <1 C63 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C66 <1 <1 <1 <1<1 11 <1 <1 <1 <1 <1 C73 <1 <1 <1 <1 2 1 <1 3 4 5 <1 C76 <1 <1 <1 <1 <1<1 <1 <1 <1 <1 <1 C80 <1 <1 <1 <1 <1 <1 1 1 2 <1 1 C81 <1 <1 <1 <1 <1 <1<1 <1 <1 <1 <1 patient 111–132 121–142 131–152 137–158 M1P1 M1P2 M1P3M1P4 C40 1 <1 <1 <1 <1 <1 <1 3 C41 <1 <1 <1 <1 <1 <1 10 <1 C43 <1 <1 <1<1 <1 <1 4 <1 C47 <1 <1 13 <1 <1 <1 <1 <1 C57 <1 <1 22 <1 <1 2 62 30 C58<1 <1 <1 <1 <1 <1 <1 <1 C63 <1 <1 <1 <1 <1 1 <1 <1 C66 <1 <1 <1 <1 31 <113 <1 C73 6 7 7 9 5 2 4 2 C76 <1 <1 <1 <1 <1 <1 15 <1 C80 1 <1 2 <1 7 27 1 C91 <1 <1 <1 <1 3 2 <1 <1

To confirm our observation that HPV16-specific memory T-cells werepresent in a large fraction of the human population we isolated thespecific CD45RO+, memory fraction of PBMC before they were subjected tostimulation with HPV16 E6 peptides. After a 4-days of stimulation aclear response to HPV16 E6 could be detected. Taken together, our datareveal the presence of CD45RO⁺ memory-type, IFNγ secreting T helpercells reactive against both HPV16 E6 peptides in approximately half ofthe healthy donors tested.

Furthermore, PBMC of two different HLA-DR1 positive blood donors werestimulated with a pool of two long 35-mer peptides that together coveredE6 109–158. PBMC of these donors proliferated specifically againstpeptide E6 121–142 and peptide E6 127–158 as well as E6 protein. A CD4+T-helper clone derived from one of these bulks was analyzed further andspecifically proliferated and produced IFNγ when stimulated with HPV16E6 peptide and HPV16 E6 protein in a HLA-DR1 restricted manner (FIG.16).

Conclusions

Through analysis of the IFNγ-responses in PBMC cultures from healthysubjects against the HPV16 E6 antigen we have demonstrated that thisprotein contains highly immunogenic peptide sequences to which strong Tcell reactivity is detected in approximately half of these donors.Subsequent testing of the CD45RO⁺ memory fraction of healthy donor PBMCrevealed that these HPV16 E6-specific IFNγ-secreting CD4⁺ T cells werepresent in the T-cell memory fraction of PBMC and thus have been primedin vivo upon encounter with HPV. A similar survey demonstrated thatHPV16 E6-specific IFNγ secreting T-cells were almost absent in HPV16+patients. Taken together, our data demonstrate that the T cellrepertoire of a majority of the healthy subjects contains particularlyhigh frequencies of memory type 1 cytokine producing T helper cellsreactive against the HPV16 E6 antigen and indicates that these areprotective against disease.

Importantly, we have not found any previous report concerning thepresence of memory T-cell responses against E2 and E6 or any of theother non-structural HPV16 proteins in healthy individuals. In fact,healthy individuals served in many cases as negative control for theculture conditions used. We have now shown, in two independent studies(example 2 and example 3), that healthy donor derived PBMC containHPV16-specific T-helper cells. The majority of the T-helper responsesare directed against specific regions in E2 and E6. The presence ofthese memory type T-helper cells show that these individuals haveencountered HPV16 infection, responded to it via the cellular T-cell armof the immune system and cleared the infection. This implies that theT-cell response to these antigens is a part of the protective responseto HPV infection. Induction or boosting of these type of responses cantherefore be a poweful tool in the protection against HPV induceddiseases.

Example 4

Material & Methods

Male C57/B16 mice (n=8 per group) were vaccinated with either 10 μg ofthe exact E1A-derived CTL epitope (SGPSNTPPEI) (SEQ. ID. NO. 1) or with30 μg of the HPLC-purified 32-amino acid long peptideRECNSSTDSCDSGPSNTPPEIHPVVRLCPIKP (SEQ. ID. NO. 2) containing this CTLepitope. Peptides were dissolved in PBS and mixed in a 1:1 ratio withIFA. Mice were vaccinated subcutaneously with peptide in a volume of 200μl at day 1. Fourteen days later mice were challenged with 0.5×10⁶ AR5tumor cells (E1A+Ras transformed mouse embryo cells). Survival of micewas monitored during a 100 day follow-up.

Results

Vaccination with synthetic peptides representing cytotoxic T lymphocyte(CTL) epitopes can lead to a protective CTL-mediated immunity againsttumors or viruses. B6 tumor cells transformed by the human adenovirusearly region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTLepitope to the immune system. CTL clones directed against either ofthese epitopes are able to eradicate established Ad5E1-induced tumors,showing that these CTL epitopes are targets of CTL that can mediatetumor regression. Protective immunity against Ad5E1-expressing tumorcells can be established by immunization with Ad5E1-transformed cellsand with an adenovirus vector containing the Ad5E1 region. Protectiveimmunity, in either case, is associated with specific CTL memory. When,however, mice were vaccinated with either the minimal peptide-epitopesequence of the E1A-derived CTL epitope or the minimal peptide-epitopesequence of the E1B-derived CTL epitope protection againstAd5A1-expressing tumors was lost.

Vaccination with a CTL epitope derived from the human adenovirus type 5E1A-region (Ad5E1A234-243), enhances rather than inhibits the growth ofAd5E1A-expressing tumors. In contrast to peptide vaccination,immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specificimmunity and prevented the outgrowth of Ad5E1A-expressing tumors. Theseresults show that immunization with synthetic peptides can lead to theelimination of anti-tumor CTL responses (Toes et al., 1996a).Furthermore, vaccinated mice s.c. with a low dose of the Ad5E1B peptidealso showed this adverse reaction. The Ad5E1B peptide was chosen becausethe CTL response against the Ad5E1B-encoded CTL epitope contributes mostto the antitumor response in B6 mice after vaccination withAd5E1-transformed cells. Ad5E1B peptide-vaccinated mice were notprotected against the outgrowth of Ad5E1-expressing tumor cells, butinstead were no longer able to reject a tumor inoculum that was rejectedby nonvaccinated mice. Moreover, the protection induced by tumor cellvaccination against Ad5E1B-expressing tumors was gone when theAd5E1B-encoded CTL epitope was injected a few days before tumorchallenge. This is associated with peptide-induced tolerance ofAd5E1B-specific CTL activity (Toes et al. 1996b).

In conclusion, immunization with synthetic peptides of the exact CTLepitope length, can also lead to CTL tolerance associated with theinability to reject tumors. The issue of tolerance or functionaldeletion of CTL by immunization with minimal epitopes is circumvented bythe use of long peptide (22–35 amino acid residues). To prove thisstatement we vaccinated C57/Bl6 mice with a 32-amino long E1A derivedpeptide that contains the 10 amino acid long CTL E1A-derived epitope,which when given as the exact peptide-epitope is involved in loss oftumor-protection. As shown in FIG. 10, control mice (n=8) that have beenvaccinated with the exact peptide-epitope SGPSNTPPEI (SEQ. ID. NO. 1),all die within 50 days after tumor-challenge. In contrast, the group ofmice vaccinated with the 32-amino acid long peptide all live at 50 daysand only one mouse is lost during the 100 day follow-up.

Conclusion

For a clinically relevant approach of immunizing subjects againstvirally infected cells or tumor cells both specific T-helper cells andCTL should be induced. We have already shown that immunization withminimal CTL epitopes results in protection against tumors in some models(Kast et al. 1991, Feltkamp et al. 1993) whereas it can also lead totolerance or functional deletion of virus- and tumor-specific CTL thatwhen otherwise induced are protective (Toes et al. 1996ab). Processingof exogenous antigens for presentation by MHC class I molecules bycross-priming as well as by other mechanisms is now widely recognizedsecond pathway of processing for presentation by MHC class I, next tothe well known endogenous route (Jondal et al., 1996, Reimann et al.1997). We have now shown that in contrast to vaccination of mice withthe exact peptide-epitope, vaccination with long peptide sequencescontaining CTL epitopes does not result in the loss of CTL that areinvolved in the protection of mice against tumors but instead result ina CTL mediated immune response that protects mice against a subsequenttumor-challenge.

Example 5

Mice and cell lines. C57BL/6 (B6, H-2^(b)) mice were obtained from IFFACredo (Paris, France). MHC class II^(−/−) B6 mice were purchased fromTaconic (USA) and CD40^(−/−) B6 mice were obtained from The JacksonLaboratory (Maine, USA). Tumor cell line TC-1 was generated bytransfection of Mouse Embryo Cells (MEC) of C57BL/6 origin by HPV 16E6/E7 and c-H-ras oncogenes. Tumor cell line 13.2 was derived from MEC(B6) transformed with adenovirus type 5 derived E1 protein in which theH-2D^(b) E1A epitope was replaced with the HPV 16 E7⁴⁹⁻⁵⁷ CTL epitope.D1 cells are long-term growth factor dependent immature splenicdendritic cells (DC) derived from C57BL/6 mice.

Peptides. The HPV16 E7-derived peptides E7₄₉₋₅₇: RAHYNIVTF (SEQ. ID. NO.3) and E7₄₄₋₇₇: GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ. ID. NO. 13)were synthesized by solid phase strategies on an automated multiplepeptide synthesizer (Abimed AMS 422, Langenfeld, Germany). The peptideswere analyzed by reverse phase HPLC for contaminants and stored at −20°C.

Tetramers and antibodies. PE-labeled H-2D^(b) epitope E7⁴⁹⁻⁵⁷(RAHYNIVTF) (SEQ. ID. NO. 3)-containing tetramers were constructed andused for the analysis of peptide-specific CTL-immunity. FITC labeledanti-CD8b.2 Ab (Ly-3.2) (clone 53-5.8), APC labeled anti-CD4 Ab (L3T4)(clone RM4-5) and PE labeled anti-IFN-γ Ab (clone XMG1.2) (BDPharMingen, San Diego, USA) were used in the various FACS procedures.

Adjuvantia. IFA (incomplete Freund's adjuvant) was obtained from DifcoLaboratories (Michigan, USA). Montanide ISA 51 was purchased from SEPPIC(Paris, France). CpG-oligodeoxynucleotides (ODN) 1826 were kindlyprovided by Dr. G. B. Lipford, Technical University of Munich (Munich,Germany). GM-CSF was obtained from PeproTech (Rocky Hill, USA). TheFGK-45 hybridoma cells producing stimulatory anti-CD40 Ab were providedby A. Rolink. MPL was kindly provided by Dr. M. Johnson, RibiImmunochem. Research (Hamilton, USA).

Immunization strategies. C57BL/6 mice were injected subcutaneously witheither 50 μg E7⁴⁹⁻⁵⁷ peptide or 150 μg E7⁴³⁻⁷⁷ 35-mer dissolved in PBSin order to achieve similar molar levels of the E7⁴⁹⁻⁵⁷ CTL epitope inboth cases. Combinations with various adjuvantia were tested. In thecase of IFA and Montanide, the dissolved peptides were emulsified in 50%of these respective substances. ODN-CpG (50 μg/mouse), MPL (10 μg/mouse)and GM-CSF (4 μg/mouse) were all dissolved in PBS and mixed with thepeptides before subcutaneous vaccination. The total injected volume was200 μl/mouse. Anti-CD40 Ab was dissolved in PBS and injected separatelyfrom the peptides intravenously on day 0, 1 and 2 at an amount of 100 μgper injection (total volume 200 μl/mouse). Either spleens were harvestedafter 10 days or, when indicated, mice were boosted with identicalvaccines 50 days after priming and spleens were harvested 10 days afterbooster immunization. In the case of the 35-mer E7⁴³⁻⁷⁷, the latterstrategy might allow the formation of memory CTL- and T helper cellsafter the first vaccination and activation of DC by HPV16 E7⁴³⁻⁷⁷specific T helper cells when the booster immunization was given. In thetherapeutic anti-tumor experiments, tumor-bearing mice were vaccinatedtwice: at the time that tumors were palpable in all mice and 14 dayslater.

T cell cultures. T cells were obtained from immunized mice by culturingspleen cells (5×10⁶ cells/well of a 24-wells plate) in complete mediumin the presence of 0.5×10⁶ E7⁴⁹⁻⁵⁷-expressing cells (tumor cell line13.2) or, when indicated, in the presence of D1 cells. Before use, theD1 cells were incubated for 16 hours with the E7⁴³⁻⁷⁷ 35-mer andsubsequently activated by adding LPS (10 μg/ml) for 6 hours and thenthoroughly washed. Complete medium consists of Iscove's ModifiedDulbecco's Medium (IMDM; BioWhittaker, Walkersville, Md., USA)supplemented with 8% FCS, 100 IU/ml penicillin, 2 mM glutamine (ICN,Aurora, Ohio, USA) and 30 μM 2-ME (Merck, Darmstadt, Germany). Cultureswere maintained at 37° C. in humidified air containing 5% CO₂. Noexogenous IL-2 was added. On day six, dead cells were removed from theculture by centrifugation over a Ficoll density gradient and remainingcells were seeded in 24-wells plates at 1×10⁶ cells/well. On day seventetramer staining or intracellular cytokine staining was performed.

Tetramer staining. Spleen cultures, stimulated with tumor cell line 13.2for seven days, were transferred at an amount of 40×10⁴ per well to96-well V-bottom microtiter plates and washed twice with PBS/BSA 0.5%.Subsequently, PE-labeled E7⁴⁹⁻⁵⁷-containing tetramer was added. After 30minutes of incubation at room temperature, cells were washed twice withPBS/BSA 0.5% and incubated with FITC-labeled anti-CD8b Ab for 30minutes. Subsequently cells were washed twice in PBS/BSA 0.5%, suspendedin PBS/BSA 0.5% containing Propidium Iodide (PI) (0.5 μg/ml) andtransferred to tubes. Cell samples were analyzed in a FACSCalibur flowcytometer (Becton Dickinson, San Jose, Calif., USA) using CellQuestsoftware. A total of 20×10⁴ events was analyzed for fluorescenceintensity. Debris was gated out using the PI staining and subsequentlythe CD8⁺ fraction was gated. Mean background tetramer-staining ofsimilarly cultured and stained cells from non-immunized control mice wasfound to be below 1 percent of CD8⁺ cells (0.94%, standard deviation0.36%). As a positive control the HPV16 E7 specific CT clone 9.5 wasused.

Intracellular cytokine staining. Spleen cultures were stimulated with D1cells pulsed with E7⁴³⁻⁷⁷ 35-mer for seven days. Subsequently, thepercentage of CD8⁺ and CD4⁺ IFN-γ producing T cells was measured asdescribed before (van der Burg et al., 2001). Notably, LPS-activatednon-pulsed D1 cells or D1 cells pulsed with E7⁴⁹⁻⁵⁷ or E7⁴³⁻⁷⁷ 35-mer (5μg/ml) were used as stimulator cells.

Results

Prime-boost Vaccinations with the E7⁴³⁻⁷⁷ 35 Amino Acid Long HPV16 E7 THelper and CTL Epitope Containing Peptide Result in a Vigorous CTLResponse.

Mice (B6) were vaccinated once with either the minimal CTL epitope HPV16E7⁴⁹⁻⁵⁷ or the HPV16 E7⁴³⁻⁷⁷ 35amino acid residue long peptide, admixedwith IFA. Ten days following vaccination spleens were harvested andstimulated in vitro for 1 week. Subsequently, the percentage of E7⁴⁹⁻⁵⁷peptide-specific CTL was determined by H2-D^(b) E7⁴⁹⁻⁵⁷ (RAHYNIVTF)(SEQ. ID. NO. 3)-tetramer staining (van der Burg et al., 2001). In bothgroups 3 out of 9 mice responded to the vaccine while generally 5percent of the CD8⁺ T cells stained with tetramers (FIGS. 11A–B),suggesting that the vaccines do equally well when injected once.

To obtain a vigorous E7⁴⁹⁻⁵⁷-specific CTL response, both vaccines wereused in a prime-boost regimen, which allowed primed T cells to formmemory T cells before the response was boosted by a second vaccination50 days later. Mice that received twice the minimal CTL epitope, showedCD8⁺ T cell responses that were comparable to that after one vaccination(FIG. 11C). In contrast, mice primed and boosted with the long peptidedisplayed a vigorous E7⁴⁹⁻⁵⁷-specific CTL response in 9/11 mice, withhigh numbers of E7⁴⁹⁻⁵⁷-specific CD8⁺ T cells (mean 19%, range 5–40%)(FIG. 11D). These experiments demonstrate that vaccination with the longpeptide in a homologous prime-boost regimen is superior to vaccinationwith the minimal CTL epitope and this indicates that the formation of Thelper cells, by the intrinsic T helper epitope present in the longpeptide, contributes considerably to the level of the CTL responses.

The Vigorous E7⁴⁹⁻⁵⁷-specific CTL Response is Dependent on MHC ClassII-restricted T Helper Cells and CD40-CD40L Interactions.

In order to demonstrate that the impressive CTL responses, detectedfollowing prime-boost vaccinations with the long E7⁴⁸⁻⁷⁷ peptide, werein fact enhanced by MHC class II-restricted E7-specific T helper cells,MHC class II^(−/−) mice were prime-boosted with the long peptidevaccine. The number of E7⁴⁹⁻⁵⁷-specific CTL detected in the MHC classII^(−/−) mice was far lower than found in B6 mice after twovaccinations, and comparable to that found after one vaccination (FIG.11E). To confirm that CD4⁺ T helper cells are already induced afterpriming, mice were vaccinated once with the long peptide in IFA.Subsequently the percentage of E7⁴³⁻⁷⁷-specific IFN?-producing CD4⁺ Tcells was measured by intracellular cytokine staining. Whereas noresponses over background were observed in naïve mice (data not shown),5 percent or more of the CD4⁺ T cells from vaccinated mice specificallyresponded upon stimulation with the long E7⁴³⁻⁷⁷ peptide (FIG. 12A).These data not only indicate that MHC class II restricted CD4⁺ T helpertype 1 responses are induced after one vaccination but also that theseE7⁴³⁻⁷⁷ peptide-specific T helper cells are required for inducingvigorous CTL responses. Professional APC, that process and present thelong peptide upon the booster immunization, are activated throughCD40-CD40L interactions by E7⁴³⁻⁷⁷peptide-specific T helper cells. Thiswill subsequently lead to enhanced CTL activation.

Single Vaccinations with the E7⁴³⁻⁷⁷ Peptide and DC-activating AgentsResult in Vigorous T Helper- and CTL Responses Towards HPV16 E7.

If the T helper-mediated activation of professional APC is important forthe observed vigorous CTL response, direct activation of DC shouldbypass a lack of T cell help and a single vaccination should besufficient to raise adequate CTL responses. To address this subject, B6mice were vaccinated once with the long peptide or the minimal E7⁴⁹⁻⁵⁷peptide in combination with various DC-activating agents. IFA andMontanide—a human grade IFA—were used as controls. As with IFA (FIGS.11A–B) no responses were observed when Montanide adjuvant wasadministered (data not shown). Furthermore, mice vaccinated with eitherthe minimal CTL epitope or the long peptide combined with anti-CD40antibody or GM-CSF displayed no or minimal CTL responses (FIGS. 13A–B).However, the combination of GM-CSF and IFA resulted in moderateE7⁴⁹⁻⁵⁷-specific CTL responses when the long peptide was administered(FIG. 13C). Mice receiving the minimal peptide mixed with MPL or ODN-CpGshowed clear-cut CTL responses in several mice (FIGS. 13D–E) althoughthe number of responders was not increased (4/9 and 4/12 micerespectively) compared to mice vaccinated with IFA (FIG. 11A). Incontrast, vaccination with the long peptide resulted in E7⁴⁹⁻⁵⁷-specificCTL responses in all mice. Moreover, the level of the CTL responsedetected was high (up to 40% of CD8⁺ T cells) in the majority ofanimals. In addition, a single vaccination with the long peptide andODN-CpG resulted in even higher numbers of E7⁴³⁻⁷⁷-specificIFNγ-producing CD4⁺ T cells (15 to 20% of CD4⁺ T-cells) when compared tothe use of IFA as adjuvant (FIG. 12B). To demonstrate that the inducedresponses were indeed T helper cell independent, MHC class II^(−/−) micewere vaccinated with the long peptide and ODN-CpG. As shown in FIG. 13F,robust CTL responses were detected after one vaccination. Theobservation that in a T helper independent setting, vaccination with thelong peptide and a DC-activating agents is superior to vaccination withthe minimal CTL epitope shows that, compared to the minimal CTL epitopethat can bind to MHC class I molecules present on all nucleated cells,the long peptide is preferentially processed and presented byprofessional antigen presenting cells (APC).

Vaccination with the 35-mer and the DC-activating Adjuvant ODN-CpG canEffectively Eradicate HPV16 Expressing Tumors.

In order to test the efficacy of the long peptide vaccine, tumor bearingmice were immunized with the E7⁴⁹⁻⁵⁷ peptide or the E7⁴³⁻⁷⁷ long peptideadmixed with ODN-CpG. Notably, the vaccination was given at the timewhen tumors were palpable (day 10–14) in all mice. A second vaccinationwas administered 14 days later in order to sustain E7-specific T helper-and CTL immunity. Mice treated with ODN-CpG alone did not displayanti-tumor activity (FIG. 14A). The majority of animals had to be killeddue to extensive tumor growth within 14 days after treatment. In bothgroups treated with peptide and ODN-CpG inhibition of tumor growth wasseen 8–12 days after treatment. Although complete eradication wasobserved in 3 out of 9 mice treated with the E7⁴⁹⁻⁵⁷ peptide and ODN-CpG(30% of the animals), others were only capable of stabilizing tumorgrowth temporarily and then died of their tumor. In contrast, 8 out of10 mice treated with the long peptide and ODN-CpG eradicated theirtumors, some of them sizing up from 200–500 mm³. In

Conclusion

We show that the HPV16 E7-specific CTL response is far more vigorousafter vaccination with a HPV16 E7 derived 35-residue long peptide thanfollowing vaccination with the minimal CTL epitope. Our data demonstratethat at least one of two independent mechanisms account for this.Firstly, the long 35-residue peptide used in this example contains botha CTL epitope and a T helper epitope. Vaccination of MHC class II−/−mice demonstrated that the interaction between APC and E7-specificT-helper cells contributed considerably to the level of the CTLresponse. Furthermore, administration of said long peptide mixed withstrong DC-activating agents was able to bypass the requirement for Tcell help. Secondly, a direct comparison of the CTL response induced bythe minimal CTL epitope (9 residues) of this example or said longpeptide vaccine combined with DC activating agents in both wild type andMHC class II−/− mice showed that vaccination with the long peptideresulted in a far better CTL response, showing that in contrast to theminimal CTL epitope, the long peptide is preferably presented byprofessional APC. Moreover, we show that vaccination with the longpeptide and strong DC activating agents result in the completeeradication of established tumors. These data provide a scientific basisfor the use of long overlapping peptides, alone or in combination withstrong DC activating agents in future human trials.

The high efficacy of our long peptide-vaccine is among other things dueto the fact that the T helper- and CTL epitope are physically linked toeach other. The potential advantage of epitope-linkage lies in theincreased chance for simultaneous presentation of both the MHC class Iand class II restricted epitopes on the surface of a single APC, therebyfacilitating the delivery of cognate T cell help to CTL priming. Adirect comparison of vaccines that used a mix of CTL and Th-epitopeswith vaccines that used physically linked, but identical, CTL andTh-epitopes demonstrated that the latter resulted in more vigorous CTLresponses (Shirai, 1994; Hiranuma, 1999; Bristol, 2000). Ourvaccinations of normal B6 and MHC class II−/− mice confirm that the useof a physically linked T-helper epitope is advantageous for thedevelopment of a strong CTL response. In addition, these experimentsdemonstrated that the interaction between E7-specific Th-cells and APCis responsible for this boost of the CTL response. Comparison of the CTLresponse induced by the minimal CTL epitope and the long peptide innormal and MHC class II−/− mice revealed another interesting property ofour long peptide vaccine. In these experiments the contribution ofE7-specific Th-cells was eliminated and only differences in physicalproperties or kinetics of the peptides are likely to play a role.Because of its size, which excluded direct binding of peptide to MHCclass I, said long peptide needed to be taken up by professional APCthat are able to process exogenously derived antigens and presentpeptides in MHC class I. We demonstrated that DC activation onlymarginally affected the outcome of the E7-specific CTL response when theminimal peptide was used whereas DC activation was a prerequisite forthe induction of a superior E7-specific CTL response by the long peptidevaccine. Together these data show that the CTL and Th-epitope present insaid long peptide are preferably presented at the surface ofprofessional APC.

Vaccination with the minimal HPV16-derived CTL epitope results in adetectable CTL response that is not improved when DC activating agentsare co-injected. This indicates that said peptide is not only presentedat the surface of activated professional APC but also in MHC class Imolecules of other nucleated cells. This is not surprising because saidpeptide can directly bind to MHC class I at the cell surface (Feltkampet al., 1993). Presentation of said CTL peptide by cells that cannotdeliver co-stimulation (e.g. non-immune cells) results in a dampening ofCTL responses and the presentation of saidE7-CTL epitope by thesenon-professional APC are, therefore, deleterious to the induction ofE7-specific CTL. Notably, we have shown previously that vaccination withthe minimal E1A-derived CTL epitope resulted in tolerance ofE1A-specific CTL that, otherwise, were protective against E1A inducedtumors (Toes 1996). Although, the possible presentation of the HPV16E7-derived CTL epitope at the surface of non-professional APC does nothinder the induction of E7-specific CTL as seen in the adenovirus tumormodel, the anti-tumor efficacy of these responses is hampered. Treatmentof established tumors by therapeutic vaccination with the minimal CTLepitope and the DC activating agent CpG is far less effective whencompared to the long peptide vaccine mixed with DC activating agents.

These results provide a scientific basis for the evaluation of thisvaccine in human trials for both prophylactic and therapeuticintervention against HPV16 induced disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the peptide E7₄₁₋₆₂ induced T-cell response thatrecognizes naturally processed antigen (Ag). PBMC from a healthyHLA-DR15, 4 and DQ6,7 positive blood donor were stimulated four timeswith peptide E7₄₁₋₆₂. The responding T-cells were tested in a 3-dayproliferation assay (a, b, c) or stimulated for 1 day in order tomeasure the production of IFNγ by ELISA (d). Responder cells (R) andautologous or MHC class II matched antigen presenting cells (A) wereincubated with the indicated Ags: recombinant HPV16 E6 protein (E6),recombinant HPV16 E7 protein (E7), recombinant HIV RT protein (RT) orpeptides derived from E6 (eg E6/81-102, peptide E6₈₁₋₁₀₂) or derivedfrom E7. Proliferation of the bulk T-cell culture (a), proliferation ofthe T-helper clone, derived from the bulk T-cell culture, and blockingof the E7 protein-specific response by adding an antibody against HLA-DR(b), proliferation of the T-helper clone when stimulated with partiallyMHC class II matched antigen presenting cells (c), and fine mapping ofthe minimal epitope by measurement of the IFNγ production per 24 h bythe T-helper clone (d).

FIG. 2 depicts a peptide E7₂₂₋₅₆ induced T-cell response that recognizesnaturally processed Ag. PBMC from a healthy HLA-DR3 and DQ2 positiveblood donor were stimulated four times with peptide E7₂₂₋₅₆.Proliferation of the bulk T-cell culture (a), specific proliferation ofthe T-helper clone, derived from the bulk T-cell culture, whenstimulated with E7₂₂₋₅₆, E7₃₁₋₅₂ and E7 protein (b), and fine mapping ofthe minimal epitope and MHC class II restriction by measurement of theIFNγ production per 24 h of the T-helper cell clone (c). (See, also,FIG. 1 and associated description).

FIG. 3 depicts a peptide E7₄₃₋₇₇ induced T-cell response that recognizesnaturally processed Ag. PBMC from a healthy HLA-DR1,3 and DQ2 positiveblood donor were stimulated four times with peptide E7₄₃₋₇₇.Proliferation of the bulk T-cell culture (a), specific proliferation ofthe T-helper cell clone against peptide E7₄₃₋₇₇ and E7 protein (b),recognition of partially matched (DR3,DQ2) peptide pulsed antigenpresenting cell (“APC”) (c) anti HLA-DR antibody blocks the E7-specificproliferation of the T-helper cell clone revealing HLA-DR3 asrestriction element (d), and specific production of IFNγ by the T-helpercell clone when stimulated with E7₄₃₋₇₇ peptide or E7 protein (e). (See,also, FIG. 1 and associated description).

FIG. 4 depicts the stimulation with influenza matrix 1 (M1)-peptides ofMACS-separated CD45RA+ (naïve) T-cells (left) and CD45RO+ (memory)T-cells (right) results in the production of IFNγ in the CD45RO+ subsetonly. The Ml protein was divided in 16 overlapping peptides. Each poolconsists of four 30 amino acid long peptides that overlap by 15 aminoacids. Tet. tox: tetanus toxoid.

FIG. 5 depicts a peptides E2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅ induced T-cellresponse that recognizes naturally processed Ag. PBMC from a healthyHLA-DR2 positive blood donor were stimulated four times with peptidesE2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅. Specific proliferation of the T-helper cellclone against peptide E2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅ and E2 protein (a),specific production of IFNγ by the T-helper cell clone when stimulatedwith E2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅ peptides (b), fine-mapping of the minimalepitope E2₃₁₆₋₃₃₀ (c) and anti HLA-DR antibody blocks the E2-specificIFNγ production of the T-helper cell clone revealing HLA-DR2 asrestriction element (d),. (See, also, FIG. 1 and associateddescription).

FIG. 6 depicts a peptides E2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅ induced T-cellresponse that recognizes naturally processed Ag. PBMC from a healthyHLA-DR2, DQ6(1) positive blood donor were stimulated four times withpeptides E2₃₀₁₋₃₃₀ and E2₃₁₆₋₃₄₅. Specific proliferation of the T-helpercell clone against peptide E2₃₀₁₋₃₃₀ and E2 protein (a), specificproduction of IFNγ by the T-helper cell clone when stimulated withE2₃₀₁₋₃₃₀ (b), fine-mapping of the minimal epitope E2₃₁₁₋₃₂₅ (c), andanti HLA-DQ antibody blocks the E2-specific IFNγ production of theT-helper cell clone revealing HLA-DQ6(1) as restriction element(d).(See, also, FIG. 1 and associated description).

FIG. 7 depicts a peptide E2₃₃₁₋₃₆₅ induced T-cell response thatrecognizes naturally processed Ag. PBMC from a healthy HLA-DR2 positiveblood donor were stimulated four times with peptide E2₃₃₁₋₃₆₅. Specificproliferation of the T-helper cell clone against peptide E2₃₃₁₋₃₆₅ andE2 protein (a), specific production of IFNγ by the T-helper cell clonewhen stimulated with E2₃₃₁₋₃₆₅ peptide (b), fine-mapping of the minimalepitope E2₃₄₆₋₃₅₅ (c) and anti HLA-DR antibody blocks the E2-specificIFNγ production of the T-helper cell clone revealing HLA-DR2 asrestriction element (d). (See, also, FIG. 1 and associated description).

FIG. 8 depicts a peptide E2₃₃₁₋₃₆₅ induced T-cell response thatrecognizes naturally processed Ag. PBMC from a healthy HLA-DR1 positiveblood donor were stimulated four times with peptide E2₃₃₁₋₃₆₅. Specificproliferation of the T-helper cell clone against peptide E2₃₃₁₋₃₆₅ andE2 protein (a), fine-mapping of the minimal epitope E2₃₅₁₋₃₆₅ (b), andanti HLA-DR antibody blocks the E2-specific IFNγ production of theT-helper cell clone revealing HLA-DR1 as restriction element (c). (See,also, FIG. 1 and associated description).

FIG. 9 depicts E2-peptide specific IFNγ-production of T-cells present inthe CD45RO+ (memory)-fraction of PBMC. The CD45RO+ memory fraction ofPBMC were isolated using MACS technology. Following separation thesememory T-cells were stimulated with indicated peptides. After 10 days ofincubation the cells were harvested, washed and then stimulated with theindicated peptides in a 96-well ELISPOT plate at 50,000 PBMC per well.Healthy blood donor derived CD45RO+ PBMC contain HPV16 E2-specificmemory T-cells as shown by the reaction to several pools of: two 30amino acid long E2 peptides (first amino acid of of first peptide andlast amino acid of second peptide are indicated at X-axis) that wereselected based on immunogenicity of these peptides, which wasestablished in previous assays (a–b), or four 30 amino acid long E2peptides (indicated are the position of the first and last amino acid ofthe sequence that is covered by these 4 peptides in the E2-protein)(d–f). MRM: memory response mix which consists of a mix of Mycobacteriumtuberculosis, Tetanus toxoid and Candida albicans antigens. (See, also,FIG. 4, and associated description).*, statistically significantresponse (p<0.05).

FIG. 10 depicts the survival of C57/B16 mice when immunized once with 30μg of the 32 amino acid long peptide (RECNSSTDSCDSGPSNTPPEIHPVVRLCPIIKP)(SEQ. ID. NO. 2) containing the minimal CTL epitope. Mice werechallenged 14 days later with 0.5 million AR5 tumor cells at theopposite flank. In contrast, mice immunized with the minimal 10 aminoacid long CTL epitope (SGPSNTPPEI) (SEQ. ID. NO. 1) were not protected.

FIGS. 11A–E. MHC class II T helper cells enhance E7⁴⁹⁻⁵⁷-specific CTLresponses following prime-boost vaccinations with the HPV16 E7⁴³⁻⁷⁷35-mer. Mice (B6) were either primed once with E7⁴⁹⁻⁵⁷ peptide (A) orE7⁴³⁻⁷⁷ 35-mer (B), or boosted 50 days later with the same peptides:E7⁴⁹⁻⁵⁷ peptide (C) or E7⁴³⁻⁷⁷ 35-mer (D). MHC class II^(−/−) mice weresimilarly primed and boosted with E7⁴³⁻⁷⁷ 35-mer (E). Peptides wereemulsified in IFA. Ten days following the final vaccination spleens wereharvested and cultures were stimulated with the E7⁴⁹⁻⁵⁷-expressing tumorcell line 13.2. Based on the forward and side scatter pattern ofisolated spleen cells in the FACS, CD8 (FL4-M) staining andH-2D^(b)-RAHYNIVTF (SEQ. ID. NO. 3)-tetramers staining spleen cells aresubjected to the analysis of CD8+ H-2D-RAHYNIVTF (SEQ. ID. NO.3)-tetramer positive T-cells. Indicated on the y-axis are the percentageof CD8+ T-cells that were H-2D^(b)-RAHYNIVTF (SEQ. ID. NO. 3)-tetramerspositive. Each column on the x-axis represents an individual mouse. Thehorizontal line indicates background responses plus standard deviationof naïve mice (1.31%).

FIGS. 12A–B. E7-specific T helper type 1 cells are induced followingvaccination with the E7⁴³⁻⁷⁷ 35-mer. Sets of 5 Mice (B6) were injectedsubcutaneously with E7⁴³⁻⁷⁷ 35-mer admixed with IFA (A) or CpG (B).Spleens were harvested after 10-days and spleen cells cultures werestimulated with D1 cells pulsed with the E7⁴³⁻⁷⁷ 35-mer. After 1 week ofin vitro stimulation the specificity of the CD4⁺ fraction of thecultures was tested by measuring the IFN-γ production of individual Tcells. White columns illustrate background IFN-γ production by CD4+T-helper cells stimulated with non-pulsed D1 cells, whereas blackcolumns represent IFN-γ production by CD4+ T-helper cells whenstimulated with E7⁴³⁻⁷⁷ 35-mer pulsed D1 cells. Each set of two columnsrepresents an individual mouse.

FIGS. 13A–F. A single vaccination of the E7⁴³⁻⁷⁷ 35-mer combined withDC-activating adjuvantia results in high CTL responses. Mice (B6) wereonly primed and not boosted with the E7⁴⁹⁻⁵⁷ peptide (depicted on theleft) or the 35-mer (depicted on the right). Anti-CD40 Ab (A), GM-CSF(B), GM-CSF plus IFA (C), MPL (D) and ODN-CpG (E) were used asadjuvants. Similarly, MHC class II^(−/−) mice were only primed and notboosted with the E7⁴³⁻⁷⁷ 35-mer and ODN-CpG (13F). Spleens wereharvested after 10-days and cultures were in vitro stimulated with theE7⁴⁹⁻⁵⁷-expressing tumor cell line 13.2 for 7 days. Subsequently, thepercentage of E7⁴⁹⁻⁵⁷-tetramer-positive CD8⁺ cells was determined byFACS-analysis (y-axis). Each column on the x-axis represents anindividual mouse. The horizontal line indicates background responsesplus standard deviation of naïve mice (1.31%).

FIG. 14 depicts the outgrowth of HPV16 E7 positive tumors in either TC-1tumor challenged mice that were vaccinated with either the minimal CTLepitope E7₄₉₋₅₇ mixed with a dendritic cell activating agent (CpG) (B;n=9 mice) or vaccinated with HPV16 E7₄₃₋₇₇ mixed mixed with a dendriticcell activating adjuvant (CpG) (C; n=10 mice) or not vaccinated (A, n=10mice) at the day that all mice had small palpable tumors (day 10). Micewere boosted 14 days later and tumor outgrowth was followed for 65 daysafter the first immunization. Note that tumor volumes started todecrease 7–10 days after immunization and they had reached volumes of200–500 mm³. Furthermore, non-immunized mice died 2–3 days after such atumor volume was reached. 6/9 mice vaccinated with the minimal epitopedied of their tumor, whereas 8/10 mice vaccinated with the long peptidecompletely eradicated their tumor.

FIG. 15 depicts the proliferation of human CD4+ T-helper clonesstimulated with peptide E2 46–75 that recognizes naturally processed Ag.PBMC from a healthy HLA-DR4 positive blood donor were stimulated fourtimes with peptides E2₄₆₋₇₅. Specific proliferation of the T-helper cellclone 47 against peptide E2 ₄₆₋₇₅ and the N-terminal E2 protein but notthe C-terminal half of the E2 protein (left) and the restriction viaHLA-DR4 (right) is shown. The peptide-specificity of T-helper clone 36that recognizes a different, yet overlapping, peptide E2₆₁₋₇₅ is shownat the bottom. (See, also, FIG. 1 and associated description).

FIG. 16 depicts a peptide E6₁₂₇₋₁₅₈ induced T-cell response thatrecognizes naturally processed Ag. PBMC from a healthy HLA-DR1 positiveblood donor were stimulated four times with peptide E6₁₂₇₋₁₅₈.Proliferation of the bulk T-cell culture (top,left) and IFNγ production(top, right). In addition, the specific proliferation (bottom, left) andIFNγ production (bottom, right) of the T-helper clone, derived from thebulk T-cell culture, and HLA-restriction when stimulated with E6₁₂₇₋₁₅₈,or E6 protein is depicted. (See, also, FIG. 1 and associateddescription).

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1. A method for inducing and/or enhancing a human papilloma virus (HPV)antigen specific T cell response comprising providing a system capableof exhibiting said response with a peptide comprising a T-helper cellepitope specific for said HPV antigen, said peptide having a length of22–45 amino acid residues, wherein the T-helper cell epitope from saidpeptide is presented at the surface of an antigen presenting cell,wherein said presentation activates said antigen presenting cell toinduce and/or enhance the HPV antigen specific T cell response.
 2. Amethod according to claim 1, wherein said peptide has a length of 22–40amino acid residues.
 3. A method according to claim 1, wherein saidpeptide further comprises a cytotoxic T lymphocyte (CTL) epitope forsaid HPV antigen.
 4. A method according to claim 1, wherein said peptidecomprises a T-helper cell epitope specific for an HPV antigen selectedfrom the group consisting of the HPV, E2, E6 and E7 proteins.
 5. Amethod according to claim 4, wherein said HPV antigen is an HPV serotype16 antigen.
 6. A method according to claim 1, further comprisingproviding said system with an adjuvant.
 7. A method according to claim4, wherein said peptide comprises a T-helper cell epitope taken from thecentral part of an HPV E7 protein as defined by amino acid positions31–77.
 8. A method according to claim 7, wherein said peptide comprisesa T-helper cell epitope taken from a region defined by amino acidspositions 35–62 of the HPV E7 protein.
 9. A method according to claim 8,wherein said peptide comprises a T-helper cell epitope selected from HPVE7 protein amino acid positions 35–50, 50–62 and 43–77.
 10. A methodaccording to claim 4, wherein said peptide is selected from a peptideconsisting of HPV E7 amino acid positions 1–22, 1–35, 11–32, 21–42,22–56, 31–52, 31–77, 41–62, 41–72, 43–77, 51–72, 61–82, 71–92, 64–98,and 77–98.
 11. A method according to claim 4, wherein said peptidecomprises a T-helper cell epitope taken from the C-terminal part of anHPV E6 protein as defined by amino acid positions 81–158.
 12. A methodaccording to claim 11, wherein said peptide comprises a T-helper cellepitope spanning a region from amino acids positions 127–142 of the HPVE6 protein.
 13. A method according to claim 4, wherein the peptide isselected from a peptide consisting of the HPV E6 amino acid positions1–22, 11–32, 19–50, 21–42, 31–52, 41–62, 51–72, 61–82, 71–92, 81–102,91–112, 101–122, 109–140, 109–143, 111–132, 121–142, 124–158, 127–158,131–152, 141–162, and 137–158.
 14. A method according to claim 4,wherein said peptide comprises T-helper cell epitope taken from a regionthat is defined by amino acid positions 31–120, 151–195, or 271–365 ofthe HPV E2 protein.
 15. A method according to claim 14, wherein thepeptide comprises an amino acid sequence spanning a region selected fromamino acid positions 51–70, 61–76, 311–325, 316–330, 346–355, and351–365 of the HPV E2 protein.
 16. A method according to claim 6,wherein said adjuvant is selected from the group consisting of anexosome, poly I: C, poly I: poly C₁₂U, monophosphoryl lipid A, a CpGcontaining nucleic acid, a CD40 ligand or anti-CD40 antibody.